Decellularised cell wall structures from fungus and use thereof as scaffold materials

ABSTRACT

Provided herein are scaffold biomaterials comprising a decellularised fungal tissue from which cellular materials and nucleic acids of the tissue are removed, the decellularised fungal tissue comprising a cellulose- or chitin-based 3-dimensional porous structure. Methods for preparing such scaffold biomaterials, as well as uses thereof as an implantable scaffold for supporting animal cell growth, for promoting tissue regeneration, for promoting angiogenesis, for a tissue replacement procedure, and/or as a structural implant for cosmetic surgery are also provided. Therapeutic treatment and/or cosmetic methods employing such scaffolds are additionally described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional under 35 U.S.C. § 121 of co-pendingU.S. Ser. No. 16/848,412 filed Apr. 14, 2020, which is a divisionalunder 35 U.S.C. § 121 of U.S. Ser. No. 16/076,990 filed Aug. 9, 2018 andissued as U.S. Pat. No. 11,045,582 on Jun. 29, 2021, which is a 35U.S.C. § 371 National Phase Entry of the International Application No.PCT/CA2017/050163 filed Feb. 10, 2017 which designates the U.S. andwhich claims benefit under 35 U.S.C. § 119(e) of U.S. Patent ApplicationNo. 62/294,671, filed on Feb. 12, 2016, the contents of each of whichare incorporated herein by reference in their entireties.

FIELD OF INVENTION

The present invention generally relates to scaffold biomaterials anduses thereof. More specifically, the present invention relates todecellularised plant or fungus tissue, and uses thereof as scaffoldbiomaterials.

BACKGROUND

The biomaterials industry is estimated to have a market value of $90Billion USD and is driven by novel materials derived from naturalsources, synthetic polymers, metals, and ceramics. These materials canform three dimensional high porosity scaffolds possessingnano/microscale structures that are biocompatible and promote the growthof living cells. There is intense interest in novel biomaterials whichsupport the invasion and proliferation of living cells for potentialapplications in tissue engineering and regenerative medicine, forexample.

Biomaterial scaffolds have applications in multiple sectors, includingdental and cosmetic surgery, clinical and medical therapies (such asregenerative medicine, wound healing, tissue engineering and repair,etc.), and research & development (including industry and academicresearch in the biomedical sciences).

Commercial biomaterials often require complicated and time consumingproduction methods, which leads to a high cost to the end user, even ifthey are not approved for human use. In addition, most commercialbiomaterials are derived from human/animal origin, resulting inpotential rejection by the body and/or adverse immune responses and/orrisk of disease transmission. The source materials can also havenegative environmental impact, and can also lead to problems withunethical sourcing. Also, some commercial biomaterials lose their shapeafter implantation, which can result in reduced success of the tissuerepair/replacement.

The development of novel biomaterials for tissue engineering strategiesis currently under intense investigation [1-3]. Biomaterials are beingdeveloped for the local delivery of therapeutic cells to target tissues[4,5], the regeneration of damaged or diseased tissues [6-9], or thereplacement of whole organs [10-15]. In their most general form,biomaterials provide a three-dimensional (3D) scaffold which attempts tomimic the in vivo cellular milieu [14,16]. Approaches have beendeveloped to engineer the mechanical [17-24], structural [25] andbiochemical properties [26-29] of these scaffolds with varyingcomplexity. As well, significant efforts are underway to ensure thatsuch implanted biomaterials are biocompatible and stimulate only minimalimmune responses. The efforts in biomaterials research is being drivenby the significant need for replacement organs and tissues. With anaging population, the gap between patients waiting for organ transplantsand available donor organs is rapidly increasing [30]. While clinicalapplications of biomaterials have been somewhat limited, physicians havesuccessfully utilized synthetic biomaterials to treat various damagedtissues and structures, such as skin, gum, cartilage, and bone [31-36].

Biomaterial scaffolds may take several forms such as powders, gels,membranes, and pastes [1,2]. Such polymer or hydrogel formulations maybe moulded or 3D-printed to produce forms that are of therapeutic value[37-39]. An alternative approach to these synthetic strategies is wholeorgan decellularization [10,12-16]. Indeed, it has been shown that it ispossible to dissociate the cells from a donated organ, leaving behindthe scaffold matrix, commonly referred as ghost organs [14]. The ghostorgans lack any of the cells from the donor and can be subsequentlycultured with cells derived from the patient or another source. Suchapproaches have already been utilized to repair and replace defectivetissues [40-42]. In the past several years, many body parts have beencreated using synthetic and decellularization approaches, including theurethra, vaginal, ear, nose, heart, kidney, bladder, and neurologicaltissues [14,38,39,43-47].

However, these approaches are not without some disadvantages [48].Synthetic techniques can involve animal products and decellularizationstrategies still involve donor tissues and organs. There has also beenintense investigation into the development of resorbable biomaterials[49]. In these cases, the aim is to provide the body with a temporary 3Dscaffold onto which healthy tissues can form. After several week ormonths, the implanted scaffold will be resorbed leaving behind acompletely natural healthy tissue [26,29,50,51]. Although this is anappealing approach, many non-resorbable biomaterials (ceramic, titanium)have been successfully employed in clinical settings and play a majorrole in numerous therapies [2,49,52-57]. Importantly, resorbablebiomaterials suffer from the fact that regenerated tissues oftencollapse and become deformed due to the loss of structure [58-62]. Forexample, for several decades, research on ear reconstruction fromengineered cartilage has shown that biomaterial implants eventuallycollapse and become deformed as the implanted scaffolds break down andresorb [63]. However, recent successful approaches have relied on theuse of resorbable collagen scaffolds embedded with permanent titaniumwire supports [53,64,65]. Therefore, the need for non-resorbable, yetbiocompatible, scaffolds persists in the field of tissue and organengineering.

Recent complementary approaches have utilized scaffolding materials thatare not derived from human organ donors or animal products, includingvarious forms of cellulose [66-77]. Nanocrystalline, nanofibrillar andbacterial cellulose constructs and hydrogels also have been studied[78-83].

An orthogonal, yet complementary, approach to organ decellularizationand synthetic cellulose strategies has also been investigated. Thesepreliminary in vitro studies investigated cellulose biomaterials fromdecellularized apple hypanthium tissue [27].

The questions of in vivo biocompatibility, alternative biomaterials, andfurther methods of biomaterials production remain. Overall, thereremains a need in the industry for alternative, additional, and/orimproved biomaterials, methods for the production thereof, and/or usesthereof.

SUMMARY OF INVENTION

It is thus an object of the invention to provide a biomaterial which maybe used as a scaffold or implant in a variety of applications which mayinclude, but are not limited to, surgical, clinical, therapeutic,cosmetic, developmental, and/or other suitable applications.

Accordingly, in certain embodiments, there is provided herein abiomaterial generated from a plant or fungi species. The biomaterial maybe modified, for example by (i) addition of a structure (i.e. otherparts of plants or fungi, or living cells), drugs, or artificialstructures (re-absorbable or not-absorbable materials); (ii)modification of its structure with mechanical or chemical procedures tomodify the original product shape or formulation to suit differentapplications; (iii) with the addition of matrices onto or into theoriginal scaffold products (such as collagen, fibronectin or any othersubstrates) to modify cell adhesion or any other beneficial elements ofcell science such as growth factors.

Biomaterials, processes for preparation and potential uses are describedin more detail below. In certain embodiments, the biomaterial may berelatively low-cost, and/or may use a relatively efficient and/or timecondensed production procedure. Also, by using complex structures asfunctional scaffolds, a wide range of possibilities may be available toproduce complex architectures. Biomaterials may have an ability tomaintain shape, may have a relatively minimal footprint (i.e. thescaffold may be nearly invisible before and/or after angiogenesis), maybe highly biocompatible, may induce rapid vascularization, and/or maygive rise to a minimal or almost non-existent immunogenic response.

In certain embodiments, the biomaterial may be derived from plants orfungi and may therefore exhibit relatively low environmental impact,and/or may be considered organic and/or biodegradable. The biomaterialmay, in certain examples, be produced from food waste, thus offering analternative route for discarded produce.

In an embodiment, there is provided herein a scaffold biomaterialcomprising a decellularised plant or fungal tissue from which cellularmaterials and nucleic acids of the tissue are removed, thedecellularised plant or fungal tissue comprising a cellulose- orchitin-based porous structure.

In another embodiment, there is provided herein a scaffold biomaterialcomprising a decellularised plant or fungal tissue from which cellularmaterials and nucleic acids of the tissue are removed, thedecellularised plant or fungal tissue comprising a cellulose- orchitin-based 3-dimensional porous structure.

In an embodiment of the scaffold biomaterials above, the decellularisedplant or fungal tissue may comprise a plant or fungal tissue which hasbeen decellularised by thermal shock, treatment with detergent, osmoticshock, lyophilisation, physical lysing, electrical disruption, orenzymatic digestion, or any combination thereof.

In another embodiment of the scaffold material or materials above, thedecellularised plant or fungal tissue may comprise a plant or fungaltissue which has been decellularised by treatment with a detergent orsurfactant. In certain embodiments, examples of detergents may include,but are not limited to, sodium dodecyl sulphate (SDS), Triton X, EDA,alkyline treatment, acid, ionic detergent, non-ionic detergents, orzwitterionic detergents, or a combination thereof.

In another embodiment of the scaffold material or materials above, thedecellularised plant or fungal tissue may comprise a plant or fungaltissue which has been decellularised by treatment with SDS.

In still another embodiment of the scaffold material or materials above,residual SDS may be removed from the decellularised plant or fungaltissue by washing with an aqueous divalent salt solution.

In yet another embodiment of the scaffold material or materials above,residual SDS may have been removed using an aqueous divalent saltsolution to precipitate/crash a salt residue containing SDS micelles outof the solution/scaffold, and a dH₂O, acetic acid, dimethylsulfoxide(DMSO), or sonication treatment may have been used to remove the saltresidue and/or SDS micelles.

In still another embodiment of the scaffold material or materials above,the divalent salt of the aqueous divalent salt solution may compriseMgCl₂ or CaCl₂.

In another embodiment of the scaffold material or materials above, theplant or fungal tissue may have been decellularised by treatment with anSDS solution of between 0.01 to 10%, for example about 0.1% to about 1%,or, for example, about 0.1% SDS or about 1% SDS, in a solvent such aswater, ethanol, or another suitable organic solvent, and the residualSDS may have been removed using an aqueous CaCl₂ solution at aconcentration of about 100 mM followed by incubation in dH₂O.

In certain embodiments, the SDS solution may be at a higherconcentration than 0.1%, which may facilitate decellularisation, and maybe accompanied by increased washing to remove residual SDS.

In yet another embodiment of the scaffold material or materials above,the decellularised plant or fungal tissue may be functionalized at atleast some free hydroxyl functional groups through acylation,alkylation, or other covalent modification, to provide a functionalizedscaffold biomaterial.

In another embodiment of the scaffold material or materials above, thedecellularised plant or fungal tissue may be processed to introducefurther architecture and/or microarchitecture and/or may befunctionalized at at least some free hydroxyl functional groups throughacylation, alkylation, or other covalent modification, to provide afunctionalized scaffold biomaterial.

In another embodiment of the scaffold material or materials above, thedecellularised plant or fungal tissue may be processed to introducemicrochannels, and/or may be functionalized with collagen, a factor forpromoting cell-specificity, a cell growth factor, or a pharmaceuticalagent, for example.

In another embodiment of the scaffold material or materials above, thedecellularised plant or fungal tissue may be functionalized withcollagen.

In yet another embodiment of the scaffold material or materials above,the plant or fungal tissue may comprise an apple hypanthium (Maluspumila) tissue, a fern (Monilophytes) tissue, a turnip (Brassica rapa)root tissue, a gingko branch tissue, a horsetail (equisetum) tissue, ahermocallis hybrid leaf tissue, a kale (Brassica oleracea) stem tissue,a conifers Douglas Fir (Pseudotsuga menziesii) tissue, a cactus fruit(pitaya) t flesh tissue, a Maculata Vinca tissue, an Aquatic Lotus(Nelumbo nucifera) tissue, a Tulip (Tulipa gesneriana) petal tissue, aPlantain (Musa paradisiaca) tissue, a broccoli (Brassica oleracea) stemtissue, a maple leaf (Acer psuedoplatanus) stem tissue, a beet (Betavulgaris) primary root tissue, a green onion (Allium cepa) tissue, aorchid (Orchidaceae) tissue, turnip (Brassica rapa) stem tissue, a leek(Allium ampeloprasum) tissue, a maple (Acer) tree branch tissue, acelery (Apium graveolens) tissue, a green onion (Allium cepa) stemtissue, a pine tissue, an aloe vera tissue, a watermelon (Citrulluslanatus var. lanatus) tissue, a Creeping Jenny (Lysimachia nummularia)tissue, a cactae tissue, a Lychnis alpina tissue, a rhubarb (Rheumrhabarbarum) tissue, a pumpkin flesh (Cucurbita pepo) tissue, a Dracena(Asparagaceae) stem tissue, a Spiderwort (Tradescantia virginiana) stemtissue, an Asparagus (Asparagus officinalis) stem tissue, a mushroom(Fungi) tissue, a fennel (Foeniculum vulgare) tissue, a rose (Rosa)tissue, a carrot (Daucus carota) tissue, or a pear (Pomaceous) tissue.

In certain embodiments, the plant or fungal tissue may comprise agenetically altered tissue prepared via direct genome modificationand/or through selective breeding to create an additional plant orfungal architecture that is configured to physically mimic a tissueand/or to functionally promote a target tissue effect. The skilledperson having regard to the teachings herein will be able to select asuitable scaffold biomaterial to suit a particular application.

In another embodiment of the scaffold material or materials above, thescaffold biomaterial may further comprise living animal cells adhered tothe cellulose- or chitin-based 3-dimensional porous structure. Inanother embodiment, the living animal cells may be mammalian cells. Inyet another embodiment, the living animal cells may be human cells.

In another embodiment, there is provided herein a method for preparing adecellularised plant or fungal tissue from which cellular materials andnucleic acids of the tissue are removed, the decellularised plant orfungal tissue comprising a cellulose- or chitin-based 3-dimensionalporous structure, said method comprising:

-   -   providing a plant or fungal tissue having a predetermined size        and shape; and    -   decellularlising the plant or fungal tissue by thermal shock,        treatment with detergent, osmotic shock, lyophilisation,        physical lysing, electrical disruption, or enzymatic digestion,        or any combination thereof,    -   thereby removing cellular materials and nucleic acids from the        plant or fungal tissue to form the decellularised plant or        fungal tissue comprising a cellulose- or chitin-based        3-dimensional porous structure.

In another embodiment of the above method, the step of decellularisingmay comprise treatment of the plant or fungal tissue with a detergent orsurfactant. In certain embodiments, examples of detergents may include,but are not limited to, sodium dodecyl sulphate (SDS), Triton X, EDA,alkyline treatment, acid, ionic detergent, non-ionic detergents, orzwitterionic detergents, or a combination thereof. In certainembodiments, the step of decellularising may comprise treatment of theplant or fungal tissue with sodium dodecyl sulphate (SDS).

In another embodiment of the method or methods above, the decellularisedplant or fungal tissue may comprise a plant or fungal tissue which hasbeen decellularised by treatment with a detergent. Examples ofdetergents may include, but are not limited to, sodium dodecyl sulphate(SDS), Triton X, EDA, alkyline treatment, acid, ionic detergent,non-ionic detergents, zwitterionic detergents, or a combination thereof.

In another embodiment of the method or methods above, the decellularisedplant or fungal tissue may comprise a plant or fungal tissue which hasbeen decellularised by treatment with SDS.

In still another embodiment of the above method or methods above,residual SDS may be removed from the decellularised plant or fungaltissue by washing with an aqueous divalent salt solution.

In another embodiment of the above method or methods, residual SDS maybe removed using an aqueous divalent salt solution to precipitate/crasha salt residue containing SDS micelles out of the solution/scaffold, anda dH₂O, acetic acid, dimethylsulfoxide (DMSO), or sonication treatmentmay be used to remove the salt residue and/or SDS micelles. In anotherembodiment, the divalent salt of the aqueous divalent salt solution maycomprise MgCl₂ or CaCl₂.

In another embodiment of method or methods above, the plant or fungaltissue may have been decellularised by treatment with an SDS solution ofbetween 0.01 to 10%, for example about 0.1% to about 1%, or, forexample, about 0.1% SDS or about 1% SDS, in a solvent such as water,ethanol, or another suitable organic solvent, and the residual SDS mayhave been removed using an aqueous CaCl₂ solution at a concentration ofabout 100 mM followed by incubation in dH₂O.

In certain embodiments, the SDS solution may be at a higherconcentration than 0.1%, which may facilitate decellularisation, and maybe accompanied by increased washing to remove residual SDS.

In another embodiment of the above method or methods, the step ofdecellularising may comprise treatment with an SDS solution of about0.1% SDS in water, and the residual SDS may be removed followingdecellularisation using an aqueous CaCl₂ solution at a concentration ofabout 100 mM, followed by incubation in dH₂O.

In another embodiment of the above method or methods, the method mayfurther comprise a step of functionalizing at least some free hydroxylfunctional groups of the decellularised plant or fungal tissue byacylation, alkylation, or other covalent modification. In certainembodiments, the hydroxyl functional groups of the decellularised plantor fungal tissue may be functionalized with collagen.

In another embodiment of the above method or methods, the method mayfurther comprise a step of processing the decellularised plant or fungaltissue to introduce further architecture and/or micro-architecture,and/or a step of functionalizing at least some free hydroxyl functionalgroups of the decellularised plant or fungal tissue by acylation,alkylation, or other covalent modification. In certain embodiments, thedecellularised plant or fungal tissue may processed to introducemicrochannels, and/or the hydroxyl functional groups of thedecellularised plant or fungal tissue may be functionalized withcollagen, a factor for promoting cell-specificity, a cell growth factor,or a pharmaceutical agent, for example.

In another embodiment of the above method or methods, the method mayfurther comprise a step of introducing living animal cells to thecellulose- or chitin-based 3-dimensional porous structure, and allowingthe living animal cells to adhere to the cellulose- or chitin-based3-dimensional porous structure. In certain embodiments, the livinganimal cells may be mammalian cells. In certain embodiments, the livinganimal cells may be human cells.

In another embodiment, there is provided herein a scaffold biomaterialcomprising a decellularised plant or fungal tissue prepared by any ofthe above methods.

In another embodiment, there is provided herein a use of any of theabove scaffold biomaterials as an implantable scaffold for supportinganimal cell growth, for promoting tissue regeneration, for promotingangiogenesis, for a tissue replacement procedure, or as a structuralimplant for cosmetic surgery.

In another embodiment, there is provided herein a use of any of theabove scaffold biomaterials as a structural implant for repair orregeneration following spinal cord injury.

In another embodiment, there is provided herein a use of any of theabove scaffold biomaterials as a structural implant for tissuereplacement surgery and/or for tissue regeneration following surgery.

In another embodiment, there is provided herein a use of any of theabove scaffold biomaterials as a structural implant for skin graftand/or skin regeneration surgery.

In another embodiment, there is provided herein a use of any of theabove scaffold biomaterials as a structural implant for regeneration ofblood vasculature in a target tissue or region.

In another embodiment, there is provided herein a use of any of theabove scaffold biomaterials as a bone replacement, bone filling, or bonegraft material, and/or for promoting bone regeneration.

In another embodiment, there is provided herein a use of any of theabove scaffold biomaterials as a tissue replacement for skin, bone,spinal cord, heart, muscle, nerve, blood vessel, or other damaged ormalformed tissue.

In another embodiment, there is provided herein a use of any of theabove scaffold biomaterials, in hydrogel form, as a vitreous humourreplacement.

In another embodiment, there is provided herein a use of any of theabove scaffold biomaterials as an artificial bursae, wherein thescaffold biomaterial forms a sac-like structure containing scaffoldbiomaterial in hydrogel form.

In another embodiment, there is provided herein a use of any of theabove scaffold biomaterials as a structural implant for cosmeticsurgery.

In yet another embodiment of any of the above use or uses, the scaffoldbiomaterial may be a scaffold biomaterial for which the decellularisedplant or fungal tissue of the scaffold biomaterial is configured tophysically mimic a tissue of the subject and/or to functionally promotea target tissue effect in the subject.

In another embodiment, there is provided herein a method for supportinganimal cell growth, for promoting tissue regeneration, for promotingangiogenesis, for replacement of a tissue, for promoting angiogenesis,or for providing a structural scaffold in a cosmetic surgery, in asubject in need thereof, said method comprising:

-   -   providing a scaffold biomaterial according to any of the        scaffold biomaterials described above; and    -   implanting the scaffold biomaterial into the subject.

In another embodiment of the above method, the scaffold biomaterial maybe implanted at the spinal cord, and promotes repair or regenerationfollowing spinal cord injury.

In another embodiment of the above method or methods, the scaffoldbiomaterial may provide a structural implant for tissue replacementand/or for tissue regeneration in the subject.

In another embodiment of the above method or methods, the scaffoldbiomaterial may provide a structural implant for skin graft and/or skinregeneration in the subject.

In another embodiment of the above method or methods, the scaffoldbiomaterial may provide a structural implant for regeneration of bloodvasculature in a target tissue or region or the subject.

In still another embodiment of the above method or methods, the scaffoldbiomaterial may provide a bone replacement, bone filling, or bone graftmaterial, and/or may promote bone regeneration, in the subject.

In another embodiment of the above method or methods, the scaffoldbiomaterial may provide a tissue replacement for skin, bone, spinalcord, heart, muscle, nerve, blood vessel, or other damaged or malformedtissue in the subject.

In still another embodiment of the above method or methods, the scaffoldbiomaterial, in hydrogel form, may provide a vitreous humour replacementin the subject.

In yet another embodiment of the above method or methods, the scaffoldbiomaterial may provide an artificial bursae in the subject, wherein thescaffold biomaterial forms a sac-like structure containing scaffoldbiomaterial in hydrogel form.

In yet another embodiment of the above method or methods, the scaffoldbiomaterial may provide a structural implant for cosmetic surgery.

In yet another embodiment of the above method or methods, the step ofproviding a scaffold biomaterial may further include:

-   -   selecting a scaffold biomaterial as described above, for which        the decellularised plant or fungal tissue of the scaffold        biomaterial is configured to physically mimic a tissue of the        subject and/or to functionally promote a target tissue effect in        the subject.

In another embodiment, there is provided herein a kit comprising ascaffold biomaterial as described above and at least one of a containeror instructions for performing a surgical or cosmetic method asdescribed above. In certain embodiment, the kit may be a surgical kit.

In another embodiment, there is provided herein a kit comprising one ormore of an SDS solution, a CaCl₂ solution, or a PBS solution, andoptionally further comprising instructions for performing a method forpreparing a decellularised plant or fungal tissue as described above.

BRIEF DESCRIPTION OF DRAWINGS

These and other features will become more apparent from the followingdescription in which reference is made to the following figures:

FIG. 1: Decellularized cellulose scaffolds. A) Phase contrast image(light microscopy technique) of cellulose cell wall structure in adecellularized apple tissue sample. The dark lines correspond todistinct cellulose structures which form a three dimensional matrix. Theoverlapping dark structures highlight the 3D porous structure of thedecellularised scaffold. B) SEM image of a similar cellulose scaffoldrevealing its three dimensional nature and large cavities, highlightingvarious depths of internal pockets that make up the scaffold. Scalebar=200 μm;

FIG. 2: Variety of structures and origins of cellulose scaffolds. Thesenew scaffolds are obtained from plants (ex: apple, asparagus, fennel)and fungi (ex: white mushroom) by employing decellularization processes;

FIG. 3: Apple scaffold implantation in a mouse model (in vivo). Twocellulose scaffolds (5×5×1 mm) were implanted subcutaneously on thedorsal section of C57BL/10 mice. The dorsal skins were then carefullyresected and fixed in 10% formalin solution at one (A) and four (B)weeks after the surgeries. Histological analyses of the implants wereconducted using haematoxylin and eosin (H&A) staining and each implantwas analysed. After a week, cell infiltration can be seen, and fullinfiltration is reached after four weeks with the presence of functionalblood vessels (angiogenesis);

FIG. 4: Scaffold footprint and full cell infiltration and angiogenesis(in vivo). A) The high porosity of the apple derived scaffold and thethin wall structure (<100 nm) can be easily observed in this picturetaken in the middle of the implant one week after the surgery. B) Fullcell infiltration and angiogenesis with functional blood vesselformation within 4 weeks post-implantation. The cellulose scaffold isinvisible and specific cellulose staining is needed to allowobservation;

FIG. 5: Fixed and stained images of cells actin cytoskeleton culturedwithin the 3D cellulose scaffold and SEM artistic images. A) NIH3T3, B)C2C12 and C) HeLa cells were cultured onto the cellulose scaffolds for 2weeks prior to staining for actin (green) and cell nuclei (blue). Theactin cytoskeleton and nucleus of mammalian cells, cultured on glass orwithin the scaffolds, were stained according to previous protocols(Guolla, Bertrand, Haase, & Pelling, 2012; Modulevsky, Tremblay,Gullekson, Bukoresthliev, & Pelling, 2012). Briefly, samples were fixedwith 3.5% paraformaldehyde and permeabilized with Triton X-100 at 37° C.Actin was stained with phalloidin conjugated to Alexa Fluor 488(Invitrogen) and nuclei were stained by labelling the DNA with DAPI(Invitrogen). Samples were then mounted in Vecta-shield (Vector Labs).NIH3T3 and C2C12 cells display characteristic actin stress fibres foundin cultured cells. HeLa cells also display characteristic actinstructures including fewer prominent stress fibres and a large amount ofcortical actin localization. The presence of stress fibers demonstratethat the mammalian cells are adhered on the surface of the cell wallscaffold and are present as in vivo. Scale bar=25 μm and applies to all.D) and E) are SEM images with artistic cell coloration treatment tohighlight cell attachment on cellulose scaffold;

FIG. 6: Cell wall architectures found in the plant and fungus kingdoms.These examples of cellulose scaffolds were resected from animals 4 weeksafter their implantation and were stained with a hematoxyline/eosinestaining. This figure shows cell wall architectures and theirrelationship to tissue function, which may guide choice of biomaterials.Cell wall architectures found in the plant and fungus kingdoms present awide variety of structures which may be similar to tissues such as bone,skin and nerves. Depending on the targeted tissue, the determination ofthe plant source of the biomaterial may be based on the plant's physicaland chemical characteristics;

FIG. 7: Examples of histological results showing cell infiltration after1, 4 and 8 weeks post-implantation (hematoxyline/eosine staining);

FIG. 8: A) Collagen deposition (blue) inside the cellulose biomaterial(white) and the observation of blood vessels (red cells are red bloodcells). B) Graph showing a quantitative representation of thepro-angiogenic property of the scaffold (observation of functional bloodvessel within 4 weeks post-implantation);

FIG. 9: Non-resorbable characteristic of the cellulose scaffold infunction of time post-implantation;

FIG. 10: Improved cell attachment and proliferation by using calciumchloride washes;

FIG. 11: Cellulose scaffold preparation. Macroscopic appearance of afreshly cut apple hypanthium tissue (A) and the translucent cellulosescaffold biomaterial post-decellularization and absent of all nativeapple cells or cell debris (B). H&E staining of cross sectioneddecellularized cellulose scaffold (C). The cell walls thickness and theabsence of native apple cells following decellularization are shown. The3D acellular and highly porous cellulose scaffold architecture isclearly revealed by scanning electron microscopy (D). Scale bar: A-B=2mm, C-D=100 μm;

FIG. 12: Cellulose scaffolds implantation and resection. Thesubcutaneous implantations of cellulose scaffolds biomaterial wereperformed on the dorsal region of a C57BL/10ScSnJ mouse model by smallskin incisions (8 mm) (A). Each implant was measured before theirimplantation for scaffold area comparison (B). Cellulose scaffolds wereresected at 1 week (D), 4 weeks (E) and 8 weeks (F) after the surgeriesand macroscopic pictures were taken (control skin in C). The changes incellulose scaffold surface area over time are presented (G). Thepre-implantation scaffold had an area of 26.30±1.98 mm². Following theimplantation, the area of the scaffold declined to 20.74±1.80 mm2 after1 week, 16.41±2.44 mm² after 4 weeks and 13.82±3.88 mm² after 8 weeks.The surface area of the cellulose scaffold has a significant decrease ofabout 12 mm² (48%) after 8 weeks implantation (*=P<0.001; n=12-14);

FIG. 13: Biocompatibility and cell infiltration. Cross sections ofrepresentative cellulose scaffolds stained with H&E and anti-CD45. Theseglobal views show the acute moderate-severe anticipated foreign bodyreaction at 1 week (A), the mild chronic immune and subsequent cleaningprocesses at 4 weeks (B) and finally, the cellulose scaffold assimilatedinto the native mouse tissue at 8 weeks (C). Higher magnificationregions of interest (D-F) allow the observation of all the cell typepopulation within biomaterial assimilation processes. At 1 week,populations of granulocytes, specifically; polymorphonuclear (PMN) andeosinophils that characterize the acute moderate to severe immuneresponse are observed, a normal reaction to implantation procedures (D).At 4 weeks, a decreased immune response can be observed (mild to lowimmune response) and the population of cells within the epidermissurrounding scaffolds now contain higher levels of monocytes andlymphocytes characterizing chronic response (E). Finally, at 8 weeks,the immune response has completely resorbed with the epidermis tissuenow appearing normal. The immune response observed with H&E staining isconfirmed using anti-CD45 antibody, well-known markers of leukocytes(G-I). The population of cells within the scaffold are now mainlymacrophages, multinucleated cells and active fibroblasts. Scale bars:A-C=1 mm, D-F=100 μm and G-I=500 μm;

FIG. 14: Extracellular matrix deposition. Cross sections ofrepresentative cellulose scaffolds stained with Masson's Trichrome(A-C). After 1 week post-implantation, the magnification of region ofinterest in (A) show the lack of collagen structures inside the collagenscaffold (D, G). As fibroblast cells start to invade the scaffold,collagen deposits inside the cellulose scaffold can be sparsely observedafter 4 weeks (E, H). Concomitant with the observation of activatedfibroblast (spindle shaped cells) inside the cellulose scaffold,collagen network is clearly visible inside the cavities after 8 weeks(F, I). Scale bars: A-C=1 mm, D-F=100 μm and G-I=20 μm. *=collagenfibers; black arrows=cellulose cell wall; white arrow=fibroblast;

FIG. 15: Vascularization and Angiogenesis. Macroscopic observations ofblood vessels directly in the surrounding tissues around the cellulosescaffold (A). Confirmation of angiogenesis within the cellulose scaffoldby the observation of multiple blood vessel cross sections in H&Estaining (B) and Masson's Trichrome staining (C) micrographs. Theangiogenesis process was also confirmed with anti-CD31 staining toidentify endothelial cells within the cellulose scaffold (D). Scalebars: A=1 mm, B=50 μm and C-D=20 μm. White arrows=blood vessels;

FIG. 16: Fixed and stained NIH3T3, C2C12 and HeLa cells cultured onnative 3D cellulose scaffolds. Specific fluorescent staining of (A)NIH3T3, (B) C2C12 and (C) HeLa mammalian cells within the nativeunmodified cellulose scaffolds. The mammalian cells and native cellulosecell wall were stained with target specific fluorescent stains revealingthe cellulose structure (red), mammalian cell membranes (green) andnuclei (blue). The cells were cultured within the decellularizedcellulose scaffolds for four weeks prior to staining and imaging. Tosimultaneously stain the cellulose scaffold and mammalian cells, wefirst fixed the samples as described above, and then washed the 4 weekcultured samples with PBS 3 times. To label the cell wall, anestablished protocol (Truernit & Haseloff, 2008) was employed. Thesamples were rinsed with water and incubated in 1% periodic acid(Sigma-Aldrich) at room temperature for 40 minutes. The tissue wasrinsed again with water and incubated in Schiff reagent (100 mM sodiummetabisulphite and 0.15 N HCl) with 100 mg/mL propidium iodide(Invitrogen) for 2 hours. The samples were then washed with PBS. Tovisualize the mammalian cells within the plant tissue, the samples wereincubated with a solution of 5 mg/mL wheat germ agglutinin (WGA) 488(Invitrogen) and 1 mg/mL Hoechst 33342 (Invitrogen) in HBSS (20 mM HEPESat pH 7.4; 120 mM NaCl; 5.3 mM KCl; 0.8 mM MgSO4; 1.8 mM CaCl₂; and 11.1mM dextrose). WGA and Hoechst 33342 are live cell dyes that label themammalian cell membrane and nucleus, respectively. The cell wallscaffolds were then transferred onto microscope slides and mounted in achloral hydrate solution (4 g chloral hydrate, 1 mL glycerol, and 2 mLwater). Slides were kept overnight at room temperature in a closedenvironment to prevent dehydration. The samples were then placed in PBSuntil ready for imaging. Clearly the mammalian cells are distributedthroughout the surface of the biomaterial. Specifically, the mammaliancells are observed to grow in colonies within the cell wall cavities.The orthogonal view (ZY plane) show the depth of the mammalian cellpenetration within the biomaterial. The green (cell membrane) and blue(nuclei) are seen deep within the biomaterial and are observed up toimaging penetrating depth of the microscope. Confocal volumes wereacquired and projected in the XY and ZY plane. The ZY orthogonal viewsdemonstrate the depth of cell proliferation within the cellulosescaffold. The top and bottom surfaces of the scaffold are indicated.Scale bars: XY=300 mm, ZY=100 mm. In D) the biomaterial was sectioned toreveal the internal structure of the biomaterial past the penetratingimaging depth restrictions of the confocal microscope. SEM image of acellulose scaffold cross section after being seeded with C2C12 cellsthat were allowed to proliferate for four weeks. The cells weredigitally colourized in order to increase contrast between the cells andcellulose structure (Scale bar: 50 mm). The internal sections wereimaged with SEM and reveal mammalian cells throughout the biomaterialand not just at the surface. Scaffolds containing mammalian cells werefirst fixed with 3.5% paraformaldehyde as presented above, and thengently washed repeatedly with PBS. The samples were then dehydratedthrough successive gradients of ethanol (50%, 70%, 95% and 100%) anddried within a lyophilizer. Samples were then gold-coated at a currentof 15 mA for 3 minutes with a Hitachi E-1010 ion sputter device. SEMimaging was conducted at voltages ranging from 2.00-10.0 kV on a JEOLJSM-7500F FESEM;

FIG. 17: Cell proliferation and viability over time. A) NIH3T3, C2C12and HeLa cells were cultured individually in cellulose n=3 scaffolds for1, 8 and 12 weeks and then imaged with confocal microscopy after beingstained with Hoechst 33342. Cells were quantified at each time pointusing ImageJ open access software (http://rsbweb.nih.gov/ij/). Anincrease in cell population is observed in all three cell types. Itshould be noted that the increase in cell count can only be a result ofproliferation as the scaffolds were only seeded with the respective celltype at the beginning of the experiment. B) After 12 weeks of culture,C2C12 cells were fixed and stained with Hoechst 33342 (blue: viablecells) and Propidium iodide (PI) (red: apoptotic/necrotic cells).Confocal volumes were acquired and projected in the XY and ZY plane andreveal that cells have proliferated throughout the structure during the12-week culture. The cells that are apoptotic/necrotic are found indeeper regions of the scaffold. The top and bottom surfaces of thescaffold are indicated. The number of live (Hoechst (+)) and dead(Hoechst/PI (+)) cells were counted and it was found that, 98% of thecells within the scaffold are viable. Data is shown for C2C12 cells, butis similar for NIH3T3 and HeLa cells (data not shown). Scale bar: B=200mm for XY and 100 mm for ZY;

FIG. 18: CaCl₂ optimization. Phase contrast images: A, C, E, G, I, K, M,O. Hoechst (nuclear stain) fluorescence images: B, D, F, H, J, L, N, P.No CaCl₂: A-D, 10 mM CaCl₂: E-H, 100 mM CaCl₂: I-L, 1000 mM CaCl₂: M-P.No cells: A, B, E, F, I, J, M, N. Cells (C2C12 myoblasts): C, D, G, H,K, L, O, P. Improved cell growth occurred at 100 mM CaCl₂ and above. Thedark spots on the cellulose in the 100 mM and 1000 mM CaCl₂ samples arecrashed out salt as evidenced by the different localization of thenuclei in the fluorescence images, and their presence in the absence ofcells. Cells were grown on the scaffolds prior to imaging. Scale bar:200 μm. This Figure shows phase contrast (A, C, E, G, I, K, M, O) andHoechst fluorescence staining (B, D, F, H, J, L, N, P) of decellularizedscaffold without any cultured cells and without CaCl₂ (A, B); of C2C12myoblasts cultured within the scaffold without CaCl₂ (C, D); of thescaffold treated with 10 mM CaCl₂ (E, F); of C2C12 myoblasts culturedwithin the scaffold treated with 10 mM CaCl₂ (G, H); of the scaffoldtreated with 100 mM CaCl₂ (I, J); of C2C12 myoblasts cultured within thescaffold treated with 100 mM CaCl₂ (K, L); of the scaffold treated with1000 mM CaCl₂ (M, N); and of C2C12 myoblasts cultured within thescaffold treated with 1000 mM CaCl₂ (O, P);

FIG. 19: Salt residue removal. 100 mM CaCl₂ was used to remove residualSDS from the cellulose scaffold. (A) CaCl₂ salt/SDS micelles crashed outonto the surface of the biomaterial; phase contrast image. (B) The saltresidue was effectively removed with dH₂O incubation. It should be notedthat sonication treatment, acetic acid incubation, and DMSO incubationyield the same result (see FIG. 20). Scale bar=200 μm;

FIG. 20: Cell growth after salt removal. The cells grew well for each ofthe salt removal treatments. dH₂O incubation: A, B; dH₂O and sonication:C, D; acetic acid incubation: E, F; and DMSO incubation: G, H. The phasecontrast images (A, C, E, G) show that the scaffold in free of saltresidue. The Hoechst (nuclear stain) fluorescence images (B, D, F, H)show substantial cell growth after 2 days of culture. Scale: 200 μm.This Figure shows phase contrast (A, C, E, G) and Hoechst fluorescencestaining (B, D, F, H) of the decellularized apple scaffolds with 2-dayC2C12 cell growth in culture washed with different salts. In A and B,the scaffolds were incubated with dH₂O. In C and D the scaffolds wereincubated with dH₂O and sonication. In E and F, the scaffolds wereincubated with acetic acid. In G and H, the scaffolds were incubatedwith DMSO;

FIG. 21: Various salts can be used for the removal of residual SDS.Different salt compounds can be used to accomplish the same task ofremoving the residual SDS from the biomaterial. PBS, KCl, CaCl₂, andMgCl₂ (all 100 mM) were used as a salt wash to clean the biomaterial.C2C12 nuclei were stained with Hoechst on decellularized apples washedwith the different salts. Each salt treatment allowed for cell growth;however, the salts with divalent cations (CaCl₂ and MgCl₂) promotedgreater cell growth. This Figure shows histological images of C2C12nuclei (2-day growth) were stained with Hoechst on decellularized applescaffolds, washed with 100 mM of PBS, KCl, CaCl₂, MgCl₂, CuSO₄, KH₂PO₄,MgSO₄, Na₂CO₃, and sodium ibuprofen. Different salt compounds may beused to accomplish the task of removing the residual SDS from thebiomaterial. PBS, KCl, CaCl₂, MgCl₂, CuSO₄, KH₂PO₄, MgSO₄, Na₂CO₃, andsodium ibuprofen (all 100 mM) were used as a salt wash to clean thebiomaterial, and remove residual SDS. Each salt treatment shown in thisfigure allowed for cell growth; however, the salts with divalent cations(CaCl₂ and MgCl₂) as well as the carbonate anion group promoted greatercell growth;

FIG. 22: The secondary wall staining of the apple scaffold and theasparagus scaffold are shown. Different elements of the cell wall can beexploited for the biomaterial. The cinnamaldehyde groups of the ligninwere stained (light purple) with Wiesner stain. The pectin and ligninwere stained with Toluidine blue O. The cellulose and β-(1-4)-glucanswere stained with Congo red;

FIG. 23: It is shown herein that native cellulose can support mammaliancells, including C2C12 myoblast, 3T3 fibroblast and human epithelialHeLa cells. However, a functional biomaterial may further be able to bechemically and mechanically tuned to suit the particular intended use.Two different techniques were used in these experiments to change thestiffness of the decellularized cellulose scaffold. Additionally, phasecontrast images demonstrate that the biomaterials still supportmammalian cell culture after chemical and physical modification. A) Thelocal mechanical elasticity of native tissue, decellularized (SDS),collagen functionalized (SDS+Coll) and glutaraldehyde (SDS+GA)cross-linked cellulose scaffolds. The native tissue and unmodifiedscaffolds do not display any significant difference in mechanicalproperties. Both the collagen functionalized and chemically cross-linkedscaffolds displayed a significant increase in elasticity compared to theDMEM scaffolds (***=p<0.001). The (B) decellularized (SDS), (C) Collagenfunctionalized (SDS+Coll) and (D) glutaraldheyde cross-linked (SDS+GA)scaffolds all supported the growth of C2C12 cells. Scale bar=200 mm;

FIG. 24: Inverse moulding techniques. Cellulose ring constructs fromdecellularized apple scaffold were cut using biopsy punches. C2C12myoblast cells were cultured on the scaffolds for 2 weeks. Thebiomaterial was fully invaded by the cells. The rings were also used incombination with temporary inverse moulding using gelatin (B), andpermanent inverse moulding using collagen (C). Both gave comparable cellgrowth to the bare cellulose scaffold (A). The C2C12 nuclei were stainedwith Hoechst (blue), the C2C12 cell membranes were stained with WGA(green), and the cellulose was stained with Schiff Reagent and propidiumiodide (red). Scale bar=1000 The first column shows C2C12 nuclei stainedwith Hoechst. The second column shows C2C12 cell membranes stained withWGA used in combination with temporary inverse moulding using gelatin.The third column shows cellulose from cultured C2C12 cells stained withSchiff Reagent and propidium iodide used in combination with permanentinverse moulding using collagen. The fourth column shows a merger of theimages in each of rows A, B and C;

FIG. 25: Cell growth and inverse moulding. Confocal imaging of C2C12cells on the native biomaterial (A), the temporary inverse mouldedbiomaterial using gelatin (B), and the permanent inverse mouldedbiomaterial using collagen (C). The xy and zy max projections are shown.The three different conditions give the same result: full invasion andhigh proliferation. The cellulose was stained with Schiff Reagent withpropidium iodide (red), and the cell nuclei were stained with Hoechst(blue). Scale: 200 μm;

FIG. 26: Cell invasion and proliferation and inverse mouldingtechniques. The cell proliferation was estimated by calculating thetotal nuclear area for each molding technique (the control is nativecellulose) (A). There was no significant difference between the nativecellulose, the gelatin moulded, and the collagen moulded samples. Thecell invasion was estimated using the ratio of the top:bottom nucleararea (B). There was no significant difference between the threeconditions. As a result, the inverse moulding did not alter the cellinvasion and proliferation in these experimental conditions;

FIG. 27: Artificial micro-architecture was created in apple derivedcellulose scaffolds. Two different micro-architectures were createdwithin the decellularized cellulose scaffolds to demonstrate thefeasibility of creating different micro-architecture with thebiomaterial for specific purposes such as increasing host cell migrationinto the cellulose scaffold. In A) a 1 mm biopsy punch was used tocreate five negative cylindrical spaces within an apple-derivedcellulose scaffold as a first example of an artificialmicro-architecture. Conversely, in B) a 3 mm biopsy punch was used tocreate a single centered negative space. Only after 4 weeks implantationincreased blood vessel formation could be observed stemming directlyfrom the artificial derived negative spaces (C and D) in both the 1 mmand 3 mm examples. In C) blood vessels are in each of four corners ofthe biomaterial suggesting the increase of vascularization within theartificial derived negative space. Similarly, in D) blood vessels can beobserved on the top of the cellulose scaffold suggesting that the bloodvessels travelled through the cellulose scaffold. Cross sections ofrepresentative cellulose scaffolds stained with haemotoxylin and eosin(H&E) (E-F);

FIG. 28: Shows pictures depicting cellulose scaffolds from varioussources, their resection and histology after 4 weeks and 8 weeks asindicated. Various plant derived cellulose scaffolds were subcutaneouslyimplanted within mice to assess biocompatibility at 4 weeks and/or 8weeks. Selective tissue of various plants were implanted for a period of4 or 8 weeks to demonstrate the biocompatibility of plant derivedcellulose and the plant architecture on in vivo host cell migration. Inall examples, cell migration and proliferation into the cellulosescaffold was observed, highlighting the biocompatibility of the plantderived cellulose scaffolds in these experiments. The subcutaneousimplantations of cellulose scaffold biomaterials were performed on thedorsal region of a C57BL/10ScSnJ mouse model by small skin incisions (8mm). Each implant was measured before their implantation for scaffoldarea comparison (first column: Cellulose Scaffold). Cellulose graftswere resected (second column: Resection) at 4 or 8 weeks as indicated.Serial 5 μm thick sections were cut, beginning at 1 mm inside thecellulose scaffold, and stained with hematoxylin-eosin (H&E) (thirdcolumn: Histology). For the evaluation of cell infiltration, micrographswere captured using Zeiss MIRAX MIDI Slide Scanner (Zeiss, Toronto,Canada) equipped with 40× objective and analysed using Pannoramic Viewer(3DHISTECH Ltd., Budapest, Hungary) and ImageJ software;

FIG. 29: Cellulose scaffolds implantation and resection. Thesubcutaneous implantations of cellulose scaffolds biomaterial wereperformed on the dorsal region of a C57BL/10ScSnJ mouse model by smallskin incisions (8 mm) (A). Each implant was measured before theirimplantation for scaffold area comparison (B). Celluose scaffolds wereresected at 1 week (D), 4 weeks (E) and 8 weeks (F) after the surgeriesand macroscopic pictures were taken (control skin in C). At each timepoint blood vessels are clearly integrated with the cellulose implantdemonstrating the biocompatibility. As well there is no acute or chronicinflammation in the tissue surrounding the implant. The changes incellulose scaffold surface area over time are presented (G). Thepre-implantation scaffold had an area of 26.30±1.98 mm². Following theimplantation, the area of the scaffold declined to 20.74±1.80 mm² after1 week, 16.41±2.44 mm² after 4 weeks and 13.82±3.88 mm² after 8 weeks.The surface area of the cellulose scaffold has a significant decrease ofabout 12 mm² (48%) after 8 weeks implantation (*=P<0.001; n=12-14);

FIG. 30: Biocompatibility and cell infiltration. Cross sections ofrepresentative cellulose scaffolds stained with H&E and anti-CD45. Theseglobal view show the acute moderate-severe anticipated foreign bodyreaction at 1 week (A), the mild chronic immune and subsequent cleaningprocesses at 4 weeks (B) and finally, the cellulose scaffold assimilatedinto the native mouse tissue at 8 weeks (C). Higher magnificationregions of interest (D-F), see inset (A-C), allow the observation of thecell type population within biomaterial assimilation processes. At 1week, we can observe populations of granulocytes, specifically;polymorphonuclear (PMN) and eosinophils that characterize the acutemoderate to severe immune response, a normal reaction to implantationprocedures (D). At 4 weeks, a decreased immune response can be observed(mild to low immune response) and the population of cells within theepidermis surrounding scaffolds now contain higher levels of monocytesand lymphocytes characterizing chronic response (E). Finally, at 8weeks, the immune response has completely resorbed with the epidermistissue now appearing normal (F). The immune response observed with H&Estaining is confirmed using anti-CD45 antibody, a well-known marker ofleukocytes (G-I). The population of cells within the scaffold are nowmainly macrophages, multinucleated cells and active fibroblasts. Scalebars: A-C=1 mm, D-F=100 μm and G-I=500 μm;

FIG. 31: Extracellular matrix deposition. Cross sections ofrepresentative cellulose scaffolds stained with Masson's Trichrome(A-C). After 1 week post-implantation, the magnification of region ofinterest in (A), see inset, show the lack of collagen structures insidethe collagen scaffold (D, G). As fibroblast cells start to invade thescaffold, collagen deposits inside the cellulose scaffold can besparsely observed after 4 weeks (E, H). Concomitant with the observationof activated fibroblast (spindle shaped cells) inside the cellulosescaffold, collagen network is clearly visible inside the cavities after8 weeks (F, I). Scale bars: A-C=1 mm, D-F=100 μm and G-I=20 μm.*=collagen fibers; black arrows=cellulose cell wall; whitearrow=fibroblast;

FIG. 32: Vascularization and Angiogenesis. Macroscopic observations ofblood vessels directly in the surrounding tissues around the cellulosescaffold (A). Confirmation of angiogenesis within the cellulose scaffoldby the observation of multiple blood vessel cross sections in H&Estaining (B) and Masson's Trichrome staining (C) micrographs. Theangiogenesis process was also confirmed with anti-CD31 staining toidentify endothelial cells within the cellulose scaffold (D). Scalebars: A=1 mm, B=50 μm and C-D=20 μm. White arrows=blood vessels;

FIG. 33: A) 2-photon confocal image of xylem structures (*) indecellularized asparagus (bar=0.1 mm), the cellulose-specific stain(red) is used to observe the fine structure within the plant. B) Phasecontrast image of a single continuous xylem microchannel (*) in plantxylem (bar=0.1 mm). C) SEM image of freeze-fractured xylem microchannel(bar=20 μm). D) Gross view of a decellularized plant plug ready forimplantation;

FIG. 34: A) Primary neurons (stained green with cell membrane dye)growing along the walls of the xylem microchannels in an decellularizedplant scaffold in-vitro. This cross-sectional image (2 μm thick) wasobtained 1 mm deep within a 3 mm long plug (bar=0.1 mm). B) H&E stain ofa subcutaneously implanted decellularized plant scaffold after 4-weeks(bar=1 mm). Inset: Cross-section of the xylem microchannels (bar=0.2mm). C) Gross view of a 3 mm decellularized plant graft (arrow)implanted into the spinal cord;

FIG. 35: A) MM axial views (i) superior and (iii) inferior to the (ii)graft (arrow). Laminectomy at (ii) T8/9 results in the loss of the nerveroots that are visible in (i) and (iii). B) After 8-weeks, the spinalcord and brain are removed. The graft (arrow) appears well integratedwith no signs of infection, calcification or fibrosis. C) Locomotorrecovery 8-weeks post-implantation was exhibited in (i-iii) coordinatedstepping and weight bearing on a treadmill (shown) and in a flat BBBarena. D) BBB scores for control (red, n=4) and graft-(blue, n=7) ratsin a flat BBB arena. E) staining reveals myelinated nerves from thespinal cord (sc), growing into the microchannels of the biomaterial (bm)(bar=200 μm). The interface is indicated with a dotted line;

FIG. 36: A global view of the entire spinal cord graft implanted in theT8-T9 region of the spine. The tissue is stained with H&E-LFB which showthe nuclei as dark purple and the myelinated tissue as light blue.Importantly, microchannels spanning the length of the entire graft canbe seen infiltrated with both host cells;

FIG. 37: Ventral sections of the surrounding transection site(top-cranial; bottom-caudal) were stained in both the control and spinalgraft implanted rats and stained for neural filament marker (NF200Green) and nuclei (Hoechst Blue). In A) the dark area represents thelocation of the biomaterial. Interestingly surrounding spinal graft,green filaments can be observed stretching in the ventral direction (redarrows). These filaments represent mature neurons within the transectedsite of the rat after 12 weeks in vivo. Conversely, within the controlB) organized neuro filaments cannot be observed indicating a lack ofmature filaments within the control transection site. Additionally, theHoechst stain reveals a significantly increased number of nuclei, and assuch cells, surrounding the spinal graft within the transection sitecompared to the control;

FIG. 38: Apple hypanthium tissue was decellularised and processed forskin grafts. C57BL/10ScSnJ mice had their dorsal skin shaved andsurgically prepped. (A) 10 mm outer diameter rubber pads were suturedonto the dorsal skin to keep the wound from closing. (B) A 5 mm diameterdecellularized apple hypanthium tissue was placed into the center of therubber pad and covered with a semi-permeable adhesive. (C) Photographswere taken after 4 days to measure the degree of host cell infiltrationduring the wound healing process;

FIG. 39: Plant derived cellulose scaffolds for bone grafts. Eachcylindrical (5 mm diameter, 1 mm thick) implants were measured prior tothe implantation for scaffold area comparison (A). Cellulose scaffoldimplants were implanted into the rat skull defects and positioned toremain within the skull defect. The skin was then positioned over thegraft and sutured so as to keep the scaffold in place. (B) The scaffoldand surrounding bone tissues were isolated 4 weeks after theimplantation and macroscopic pictures were taken (C). The isolatedtissue was then decalcified and processed/embedded in paraffin. Serial 5μm thick sections were cut, beginning at 1 mm inside the cellulosescaffold, and stained with hematoxylin-eosin (H&E) (D). For theevaluation of bone regeneration, micrographs were captured using ZeissMIRAX MIDI Slide Scanner (Zeiss, Toronto, Canada) equipped with 40×objective and analysed using Pannoramic Viewer (3DHISTECH Ltd.,Budapest, Hungary) and ImageJ software;

FIG. 40: Different cellulose formulations, physical properties andfunctionalization. Cellulose may be used as a block (A) with differentshapes or dehydrated and ground into a powder form that may then berehydrated to a desired consistency to produce a gel (C, D) or a paste(E, F). If the cellulose contains carboxymethylcellulose, it may easilybe crosslinked with citric acid and heat (B). Cellulose sourced fromdifferent plants may also be combined, mixed, cross-liked etc.; and

FIG. 41: This figure shows (A) a graph showing the survival rate of mice(n=190) and rats (n=12) following the implantation of the biomaterial(from various sources) at 1 week, 4 weeks and 8 weeks post-implantation.(B) This figure shows the rate of biomaterial rejection at these sametime points as in (A).

DETAILED DESCRIPTION

Described herein are scaffold biomaterials comprising a decellularisedplant or fungal tissue from which cellular materials and nucleic acidsof the tissue are removed, the decellularised plant or fungal tissuecomprising a cellulose- or chitin-based porous structure. Methods forpreparing such scaffold biomaterials, as well as uses thereof as animplantable scaffold for supporting animal cell growth, for promotingtissue regeneration, for promoting angiogenesis, for a tissuereplacement procedure, for promoting angiogenesis, and/or as astructural implant for cosmetic surgery are also provided. Therapeutictreatment and/or cosmetic methods employing such scaffolds areadditionally described, as well as other applications which may includeveterinary applications, for example. It will be appreciated thatembodiments and examples are provided for illustrative purposes intendedfor those skilled in the art, and are not meant to be limiting in anyway.

In certain embodiments, there is described herein biomaterials which mayhave applications in biomedical laboratory research and/or clinicalregenerative medicine, for example. Such biomaterials may be effectiveas scaffolds which may be used as investigative tools forindustrial/academic biomedical researchers, for biomedical implants, insensing devices and pharmaceutical delivery vehicles, and/or in othersuitable applications in which scaffolds may be used.

In certain embodiments, the biomaterials described herein may be derivedfrom cell wall architectures found in the plant and fungus kingdoms tocreate complex 3D scaffolds which may promote cell infiltration, cellgrowth, angiogenesis, tissue repair, and/or tissue reconstruction, etc.(see, for example, FIG. 1). As will be understood, biomaterials asdescribed herein may be produced from any suitable part of plant orfungal organisms, including, for example, seed, root, bark, leaf, stem,fruit, pulp, core, and may, in certain embodiments, be produced withdifferent shapes (such as sheets, vessels, blocks, cannulation, aerationholes, etc.) or formulations (including, for example, pastes, particles,blocks, etc.) (see, for example, FIG. 2). Biomaterials may comprise, forexample, substances such as cellulose, chitin and/or any other suitablebiochemicals/biopolymers which are naturally found in these organisms.

In certain embodiments, resulting scaffolds may also be: chemicallymodified to introduce custom surface chemistry; cut as solid blocks,injectable/extrudable pastes, and/or slurries; and/or may offer a rangeof architectural possibilities on the scale of micrometers tocentimeters, which may replace/mimic several kinds of living tissueenvironments.

As described herein, the use of such plant/fungus-derived biomaterialmay result in a high porosity scaffold which may have notably thin walls(<100 nm) (see, for example, FIG. 1). This may, in certain embodiments,provide a minimal footprint of the scaffold material (i.e. when fullyinvaded by living cells, the cell to scaffold volume ratio may benotably high).

In certain embodiments, scaffold biomaterials as described herein may bebiocompatible. As described in further detail below, followingsubcutaneous implantation of example scaffold biomaterials in a mousemodel, full cell infiltration and angiogenesis with functional bloodvessel formation was observed within 4 weeks post-implantation (see, forexample, FIGS. 3 and 4). As also described in the experiments detailedhereinbelow, when scaffolds were implanted in vivo, the minimumfootprint promoted cell infiltration, angiogenesis and tissue repair,and only a minimal inflammatory response (mainly produced by the surgeryitself rather than the scaffold) was observed under the conditionstested.

Experiments described herein below indicate that plant/fungus derivedbiomaterials as described herein were fully biocompatible in vivo underthe conditions tested. They were also fully compatible with in vitrostudies as shown in FIG. 5, and in Modulevsky, D. J., Lefebvre, C.,Haase, K., Al-Rekabi, Z. and Pelling, A. E. “Apple Derived CelluloseScaffolds for 3D Mammalian Cell Culture.” Plos One, 9, e97835 (2014)(herein incorporated by reference).

In certain embodiments, unlike many commercial biomaterials,plant/fungus derived biomaterials as described herein may benon-resorbable or poorly resorbable (ie. they will not substantiallybreakdown and be absorbed by the body). The non-resorbablecharacteristic of these scaffolds may offer certain benefits. Forexample, in certain embodiments, biomaterials described herein may beresistant to shape change, and/or may hold their intended geometry overlong periods of time. In certain embodiments, since they may have aminimal footprint compared to certain other products, they may beconsidered effectively invisible to the body, eliciting almost no immuneresponse. When resorbable biomaterials break down, their by-productsoften illicit an adverse immune response, as well as induce oxidativestress and result in an increase of pH in the recovering tissue, whichmay be avoided by using a non-resorbable biomaterial.

As will be understood, unless otherwise indicated, themeaning/definition of plant and fungi kingdoms used herein is based onthe Cavalier-Smith classification (1998).

Scaffold Biomaterials

In an embodiment, there is provided herein a scaffold biomaterialcomprising a decellularised plant or fungal tissue from which cellularmaterials and nucleic acids of the tissue are removed, thedecellularised plant or fungal tissue comprising a cellulose- orchitin-based 3-dimensional porous structure. As will be understood, incertain embodiments, a scaffold biomaterial may comprise a foreignmaterial to the host which may provide an underlying architecture,support and/or infrastructure for host cells to infiltrate, invade,and/or proliferate.

In certain embodiments, scaffold biomaterials may comprise asubstantially solid form, a block or other rigid shape, may bedehydrated and ground into a powdered or particle form, may be in across-linked form (particularly where the scaffold biomaterial comprisesa cellulose-based tissue which contains carboxymethylcellulose, whichmay easily be crosslinked with citric acid and heat), or may be in a gelor paste form. Such gels or pastes may be produced, for example, byrehydrating a powdered form of the tissue to a desired consistency toproduce a gel or a paste. Additionally, in certain embodiments,compression molding may be employed to generate sheets of cellulosebased biomaterials, optionally with various additives to enhancecrosslinking. Such additives may include, but are not limited to, oxalicacid, malonic acid, succinic acid, malic acid or citric acid which maybe either added to the pulp or sprayed together with sodium dihydrogenphosphate as a catalyst.

As will be understood, decellularised plant or fungal tissue maycomprise any suitable biomaterial derived or produced from a suitableplant or fungal derivative or direct tissue sample. In certainembodiments, such materials, which may comprise an underlyingarchitecture and/or mesh support structure, may result from a suitablecombined or single method to remove, lyse, or enzymatically processnative cells from either a plant or fungal tissue.

In certain embodiments of the scaffold material or materials above, theplant or fungal tissue may comprise an apple hypanthium (Malus pumila)tissue, a fern (Monilophytes) tissue, a turnip (Brassica rapa) roottissue, a gingko branch tissue, a horsetail (equisetum) tissue, ahermocallis hybrid leaf tissue, a kale (Brassica oleracea) stem tissue,a conifers Douglas Fir (Pseudotsuga menziesii) tissue, a cactus fruit(pitaya) flesh tissue, a Maculata Vinca tissue, an Aquatic Lotus(Nelumbo nucifera) tissue, a Tulip (Tulipa gesneriana) petal tissue, aPlantain (Musa paradisiaca) tissue, a broccoli (Brassica oleracea) stemtissue, a maple leaf (Acer psuedoplatanus) stem tissue, a beet (Betavulgaris) primary root tissue, a green onion (Allium cepa) tissue, aorchid (Orchidaceae) tissue, turnip (Brassica rapa) stem tissue, a leek(Allium ampeloprasum) tissue, a maple (Acer) tree branch tissue, acelery (Apium graveolens) tissue, a green onion (Allium cepa) stemtissue, a pine tissue, an aloe vera tissue, a watermelon (Citrulluslanatus var. lanatus) tissue, a Creeping Jenny (Lysimachia nummularia)tissue, a cactae tissue, a Lychnis alpina tissue, a rhubarb (Rheumrhabarbarum) tissue, a pumpkin flesh (Cucurbita pepo) tissue, a Dracena(Asparagaceae) stem tissue, a Spiderwort (Tradescantia virginiana) stemtissue, an Asparagus (Asparagus officinalis) stem tissue, a mushroom(Fungi) tissue, a fennel (Foeniculum vulgare) tissue, a rose (Rosa)tissue, a carrot (Daucus carota) tissue, or a pear (Pomaceous) tissue.

In certain embodiments, the plant or fungal tissue may be geneticallyaltered via direct genome modification or through selective breeding, tocreate an additional plant or fungal architecture which may beconfigured to physically mimic a tissue and/or to functionally promote atarget tissue effect. The skilled person having regard to the teachingsherein will be able to select a suitable scaffold biomaterial to suit aparticular application.

In certain embodiments, a suitable tissue may be selected for aparticular application based on, for example, physical characteristicssuch as size, structure (porous/tubular), stiffness, strength, hardnessand/or ductility, which may be measured and matched to a particularapplication. Moreover, chemical properties such as reactivity,coordination number, enthalpy of formation, heat of combustion,stability, toxicity, and/or types of bonds may also be considered forselection to suit a particular application. Such characteristics(physical and chemical) may also be directly modified before or afterdecellularization and/or functionalization to respond to the specificapplication. Furthermore, in certain embodiments, cellulose may besourced from different plants and may be combined and mixed, cross-likedetc. using chemistry outlined hereinbelow.

In certain embodiments, the scaffold biomaterial may be a scaffoldbiomaterial for which the decellularised plant or fungal tissue of thescaffold biomaterial is configured to physically mimic a tissue of thesubject and/or to functionally promote a target tissue effect in thesubject. Methods of using such scaffold biomaterials as are describedherein may, in certain embodiments, include a step of selecting ascaffold biomaterial as described herein for which the decellularisedplant or fungal tissue of the scaffold biomaterial is configured tophysically mimic a tissue of the subject and/or to functionally promotea target tissue effect in the subject. The skilled person having regardto the teachings herein will be able to select a suitable scaffoldbiomaterial to suit a particular application.

By way of non-limiting example, FIG. 6 provides some examples ofscaffold biomaterials demonstrating histological cell wall architecturesand corresponding relationships to certain tissues/tissue functions,which may, in certain embodiments, be used to guide selection ofscaffold biomaterial to suit particular application(s). As will beunderstood, cell wall architectures found in the plant and funguskingdoms present a wide variety of structures which may be similar totissues such as bone, skin and nerves. Depending on the targeted tissue,the determination of the plant or fungal source of the biomaterial maybe based on the plant's physical and/or chemical characteristics, and/orthe physical and/or chemical characteristics of the generated scaffoldbiomaterial.

As will be understood, cellular materials and nucleic acids may includeintracellular contents such as cellular organelles (e.g. chloroplasts,mitochondria), cellular nuclei, cellular nucleic acids, and cellularproteins. These may be substantially removed, partially removed, orfully removed from the scaffold biomaterial. It will recognized thattrace amounts of such components may still be present in thedecellularised plant or fungal tissues described herein.

As will be understood, in certain embodiments, a 3-dimensional (3D)porous structure may include a suitable structure which provides anunderlying architecture, support, and/or infrastructure for foreigncells to infiltrate, invade and/or proliferate within while providing aconstant supply of media/nutrients via passive diffusion.

Various methods may be used for producing scaffold biomaterials asdescribed herein. By way of example, in certain embodiments of thescaffold biomaterials above, the decellularised plant or fungal tissuemay comprise a plant or fungal tissue which has been decellularised bythermal shock, treatment with detergent (e.g. SDS, Triton X, EDA,alkyline treatment, acid, ionic detergent, non-ionic detergents, andzwitterionic detergents), osmotic shock, lyophilisation, physical lysing(e.g. hydrostatic pressure), electrical disruption (e.g. non thermalirreversible electroporation), or enzymatic digestion, or anycombination thereof. In certain embodiments, biomaterials as describedherein may be obtained from plants and/or fungi by employingdecellularization processes which may comprise any of several approaches(either individually or in combination) including, but not limited to,thermal shock (for example, rapid freeze thaw), chemical treatment (forexample, detergents), osmotic shock (for example, distilled water),lyophilisation, physical lysing (for example, pressure treatment),electrical disruption and/or enzymatic digestion.

In certain embodiments, the decellularised plant or fungal tissue maycomprise a plant or fungal tissue which has been decellularised bytreatment with a detergent or surfactant. Examples of detergents mayinclude, but are not limited to sodium dodecyl sulphate (SDS), Triton X,EDA, alkyline treatment, acid, ionic detergent, non-ionic detergents,and zwitterionic detergents.

In still further embodiments, the decellularised plant or fungal tissuemay comprise a plant or fungal tissue which has been decellularised bytreatment with SDS.

In still another embodiment, residual SDS may be removed from thedecellularised plant or fungal tissue by washing with an aqueousdivalent salt solution. The aqueous divalent salt solution may be usedto precipitate/crash a salt residue containing SDS micelles out of thesolution/scaffold, and a dH₂O, acetic acid or dimethylsulfoxide (DMSO)treatment, or sonication, may have been used to remove the salt residueor SDS micelles.

In certain embodiments, the divalent salt of the aqueous divalent saltsolution may comprise, for example, MgCl₂ or CaCl₂.

In another embodiment, the plant or fungal tissue may have beendecellularised by treatment with an SDS solution of between 0.01 to 10%,for example about 0.1% to about 1%, or, for example, about 0.1% SDS orabout 1% SDS, in a solvent such as water, ethanol, or another suitableorganic solvent, and the residual SDS may have been removed using anaqueous CaCl₂ solution at a concentration of about 100 mM followed byincubation in dH₂O.

In certain embodiments, the SDS solution may be at a higherconcentration than 0.1%, which may facilitate decellularisation, and maybe accompanied by increased washing to remove residual SDS.

In particular embodiments, the plant or fungal tissue may have beendecellularised by treatment with an SDS solution of about 0.1% SDS inwater, and the residual SDS may have been removed using an aqueous CaCl₂solution at a concentration of about 100 mM followed by incubation indH₂O.

Examples of experimental protocols for the preparation of biomaterialsas described herein are provided in further detail in the “ScaffoldBiomaterial Preparation Methods” section below, and in Example 1.

In yet another embodiment of the scaffold material or materials above,the decellularised plant or fungal tissue may be functionalized at atleast some free hydroxyl functional groups through acylation,alkylation, or other covalent modification, to provide a functionalizedscaffold biomaterial. In certain embodiments, the decellularised plantor fungal tissue may be functionalized with collagen, for example.

In another embodiment of the scaffold material or materials above, thescaffold biomaterial may further comprise living animal cells adhered tothe cellulose- or chitin-based 3-dimensional porous structure. Inanother embodiment, the living animal cells may be mammalian cells. Inyet another embodiment, the living animal cells may be human cells.

Scaffold Biomaterial Preparation Methods

In an embodiment, there is provided herein a method for preparing adecellularised plant or fungal tissue from which cellular materials andnucleic acids of the tissue are removed, the decellularised plant orfungal tissue comprising a cellulose- or chitin-based 3-dimensionalporous structure, said method comprising:

-   -   providing a plant or fungal tissue having a predetermined size        and shape; and    -   decellularlising the plant or fungal tissue by thermal shock,        treatment with detergent, osmotic shock, lyophilisation,        physical lysing, electrical disruption, or enzymatic digestion,        or any combination thereof,    -   thereby removing cellular materials and nucleic acids from the        plant or fungal tissue to form the decellularised plant or        fungal tissue comprising a cellulose- or chitin-based        3-dimensional porous structure.

In certain embodiments, the step of decellularising the plant or fungaltissue may comprise decellularisation by treatment with a detergent.Examples of detergents may include, but are not limited to, sodiumdodecyl sulphate (SDS), Triton X, EDA, alkyline treatment, acid, ionicdetergent, non-ionic detergents, and zwitterionic detergents.

In till further embodiments, the step of decellularising the plant orfungal tissue may comprise a plant or fungal tissue which has beendecellularised by treatment with SDS.

In still another embodiment, the step of decellularising the plant orfungal tissue, residual SDS may be removed from the decellularised plantor fungal tissue by washing with an aqueous divalent salt solution. Theaqueous divalent salt solution is used to precipitate/crash a saltresidue containing SDS micelles out of the scaffold, and a dH₂O, aceticacid or dimethylsulfoxide (DMSO) treatment or sonication, may have beenused to remove the salt residue or SDS micelles. The divalent salt ofthe aqueous divalent salt solution may comprise, for example, MgCl₂ orCaCl₂.

In a particular embodiment, the step of decellularising may comprisetreatment with an SDS solution of about 0.1% SDS in water, and theresidual SDS may be removed following decellularisation using an aqueousCaCl₂ solution at a concentration of about 100 mM, followed byincubation in dH₂O.

In another embodiment of the above method or methods, the method mayfurther comprise a step of functionalizing at least some free hydroxylfunctional groups of the decellularised plant or fungal tissue byacylation, alkylation, or other covalent modification. In certainembodiments, the hydroxyl functional groups of the decellularised plantor fungal tissue may be functionalized with collagen.

In another embodiment of the above method or methods, the method mayfurther comprise a step of introducing living animal cells to thecellulose- or chitin-based 3-dimensional porous structure, and allowingthe living animal cells to adhere to the cellulose- or chitin-based3-dimensional porous structure. In certain embodiments, the livinganimal cells may be mammalian cells. In certain embodiments, the livinganimal cells may be human cells.

Scaffold Biomaterial Applications

In certain embodiments, biomaterials as described herein may haveapplications in biomedical laboratory research and/or clinicalregenerative medicine in human and/or veterinary applications, forexample. Such biomaterials may be effective as scaffolds which may beused as investigative tools for industrial/academic biomedicalresearchers, for biomedical implants, in sensing devices andpharmaceutical delivery vehicles, and/or in other suitable applicationsin which scaffolds may be used.

In certain embodiments, scaffold biomaterials as described herein may beused as implantable scaffolds for supporting animal cell growth, forpromoting tissue regeneration, for promoting angiogenesis, for a tissuereplacement procedure, or as a structural implant for cosmetic surgery.

In certain embodiments, scaffold biomaterials as described herein may beused as a structural implant for repair or regeneration following spinalcord injury; as a structural implant for tissue replacement surgeryand/or for tissue regeneration following surgery; as a structuralimplant for skin graft and/or skin regeneration surgery; as a structuralimplant for regeneration of blood vasculature in a target tissue orregion; as a bone replacement, bone filling, or bone graft material,and/or for promoting bone regeneration; as a tissue replacement forskin, bone, spinal cord, heart, muscle, nerve, blood vessel, or otherdamaged or malformed tissue; as a vitreous humour replacement (inhydrogel form); as an artificial bursae, wherein the scaffoldbiomaterial forms a sac-like structure containing scaffold biomaterialin hydrogel form; and/or as a structural implant for cosmetic surgery,for example.

In certain embodiments, scaffold biomaterials as described herein may beused as breast implants. The scaffold may thus be formulated to matchmammary glands/tissues found in human breast and then used as a fillingmaterial for breast implants, for example.

In certain other embodiments, scaffold biomaterials as described hereinmay be used as cartilage replacements: The scaffold may thus beformulated and designed to mimic cartilage tissues and used to replacecertain body parts, such as ears and noses.

In certain embodiments, scaffold biomaterials as described herein may beused as skin grafts. The cellulose scaffold may be used as skin graft toprotect, repair and/or regenerate skin (epithelial/endothelial)following skin surgeries (ex: gum, etc.) or injury events (ex: burns,etc.). It may, in certain embodiments, be used to protect the damagedtissues against external infections and/or to directly regenerate thetissues.

In certain embodiments, scaffold biomaterials as described herein may beused for regeneration of blood vasculature. The wide range of cellulosestructures available may allow for the artificial production of bloodvessel-like structures, and/or may provide conditions suitable forangiogenesis (natural blood vessel formation).

In another embodiment, scaffold biomaterials as described herein may beused for bone replacement or bone filling. The cellulose scaffold maythus be formulated and designed to mimic bone tissues, and then used toreplace bones and bone parts such as in dentistry, skull bone, fracturedbones, hip replacement (bone or filling agent for prosthetics, etc.)and/or other such applications.

In certain embodiments, scaffold biomaterials as described herein may beused as simple or complex tissues. By way of example, scaffolds may beused to replace simple (skin, bone) or complex (spinal cord, heart,muscle, nerves, blood vessels, etc.) tissues following accident,malformation, esthetic, injury, or other damage to the tissue.

In other embodiments, scaffold biomaterials as described herein may beused as vitreous humour material. By way of example, cellulose scaffoldsin hydrogel form are a translucent gel. The consistency and clarity maybe tuned to match that of native vitreous humour.

In certain embodiments, scaffold biomaterials as described herein may beused as bursae. Artificial bursae, and their corresponding fluid, may bemade from biomaterials described herein. The bursae may be created fromthe solid cellulose, whereas the fluid may be formed from cellulosehydrogel, for example.

In certain embodiments, there are provided herein methods for supportinganimal cell growth, for promoting tissue regeneration, for promotingangiogenesis, for replacement of a tissue, or for providing a structuralscaffold in a cosmetic surgery, in a subject in need thereof, saidmethods comprising:

-   -   providing a scaffold biomaterial according to any of the        scaffold biomaterials described above; and    -   implanting the scaffold biomaterial into the subject.

In certain embodiments, the scaffold biomaterial may be implanted at thespinal cord, and promotes repair or regeneration following spinal cordinjury; may provide a structural implant for tissue replacement and/orfor tissue regeneration in the subject; may provide a structural implantfor skin graft and/or skin regeneration in the subject; may provide astructural implant for regeneration of blood vasculature in a targettissue or region or the subject; may provide a bone replacement, bonefilling, or bone graft material, and/or may promote bone regeneration,in the subject; may provide a tissue replacement for skin, bone, heart,muscle, nerve, blood vessel, or other damaged or malformed tissue in thesubject; may provide a vitreous humour replacement in the subject (whenin hydrogel form); may provide an artificial bursae in the subject,wherein the scaffold biomaterial forms a sac-like structure containingscaffold biomaterial in hydrogel form; and/or may provide a structuralimplant for cosmetic surgery.

In certain embodiments, the scaffold biomaterial may be implanted at thespinal cord, and may promote repair and/or regeneration following acuteand/or chronic spinal cord injury in the central and/or peripheralnervous system.

Example 1—Experimental Protocol Examples for Scaffold BiomaterialProduction

In this Example, two experimental protocols are described for preparingscaffold biomaterials as described herein from an apple hypanthiumtissue (Malus pumila). It will be understood that these protocols areprovided as illustrative and non-limiting examples intended for theperson of skill in the art. The skilled person having regard to theteachings herein will be aware of various modifications, additions,substitutions, and/or other changes which may be made to these exemplaryprotocols.

The initial experimental protocol described below was successfully usedfor preparing scaffold biomaterials. This protocol, however, took manyweeks to provide full cell infiltration under the conditions tested. Amodified protocol was, therefore, subsequently developed, which includesthe use of a calcium chloride wash (CaCl₂), which gave similar resultsto scaffold biomaterials prepared by the first protocol, but within aweek (see FIGS. 9 and 10).

Initial Protocol for In Vivo (Animal Model) Studies:

-   -   1. Cut apples slices to desired shape and size        -   a. Cut the apple in half        -   b. Half the apple is submerged in PBS cut face down        -   c. Adjust the mandolin slicer to get an appropriate            thickness (in this example, 1.2 mm)        -   d. Take a uniform slice with no visible apple core and place            it on the metric cutting board        -   e. Cut one side of the apple away for further processing            into squares and keep the other piece in PBS        -   f. Use the guidelines (5 mm×5 mm) to cut the apple tissue            into the squares.        -   g. Place the cut slices into the 1.5 mL micro-centrifuge            tubes        -   h. Measure the cut side of the unused slice at least 10× and            record in lab book    -   2. Add 1 mL of 0.1% SDS (in autoclaved dH₂O) and incubate on the        shaker for 2 days (room temperature) at 180 RPM RT        -   a. Check to see if the apple squares are not floating.        -   b. Continue SDS treatment if apples still floating    -   3. Take the processed apples in the micro centrifuge tubes into        the biosafety cabinet.    -   4. Remove the 0.1% SDS solution (room temperature) with the        Pasteur pipette    -   5. Wash the apple slices 4 times with autoclaved PBS (room        temperature)        -   a. During the wash try to place the Pasteur pipette as close            to the apple as possible without touching it. This is to try            to get water to flow through the apple tissue.        -   b. When there is no liquid left in the tube continue to use            the Pasteur pipette to draw liquid solution from the apple        -   c. As you do more washes the amount of “soapy foam” residue            seen being drawn through the pipette should decrease        -   d. Do not stop washing until you see no “soapy foam” being            drawn from the apple        -   e. The apple should also not be floating    -   6. Set the desired samples opposite to Sterile micro centrifuge        tubes    -   7. Remove the last PBS wash from the micro centrifuge tubes and        replace with 70% ethanol.    -   8. Leave in 70% ethanol for 30 mins-1 hour    -   9. Remove the 70% ethanol    -   10. Continue washing the apple slice with sterilized PBS with        the same technique as previously mentioned.        -   a. Make sure you change pasteur pipettes    -   11. Continue washing until the apple slices stop floating (at        least 4 times) with PBS    -   12. Remove the PBS and replace with 1% Penicillin/Streptomycin        PBS    -   13. Implant into animal model.

Modified Protocol for In Vivo Studies:

-   -   1. Cut apples slices to desired shape and size        -   a. Cut the apple in half        -   b. Half the apple is submerged in PBS cut face down        -   c. Adjust the mandolin slicer to get an appropriate            thickness (in this example, 1.2 mm)        -   d. Take a uniform slice with no visible apple core and place            it on the metric cutting board        -   e. Cut one side of the apple away for further processing            into squares and keep the other piece in PBS        -   f. Use the guidelines (5 mm×5 mm) to cut the apple tissue            into the squares.        -   g. Place the cut slices into the 1.5 mL micro-centrifuge            tubes        -   h. Measure the cut side of the unused slice at least 10× and            record in lab book    -   2. Add 1 mL of 0.1% SDS (in autoclaved dH₂O) and incubate on the        shaker for 2 days (room temperature) at 180 RPM RT        -   a. Check to see if the apple squares are not floating.        -   b. Continue SDS treatment if apples still floating    -   3. Take the processed apples in the micro centrifuge tubes into        the biosafety cabinet.    -   4. Remove the 0.1% SDS solution (room temperature) with the        Pasteur pipette    -   5. Wash the apple slices 4 times with autoclaved dH₂O (room        temperature)        -   a. During the wash try to place the Pasteur pipette as close            to the apple as possible without touching it. This is to try            to get water to flow through it.        -   b. When there is no liquid left in the tube continue to use            the Pasteur pipette to draw liquid from the apple        -   c. As you do more washes the amount of “soapy foam” residue            seen being drawn through the pipette should decrease        -   d. Do not stop washing until you see no “soapy foam” being            drawn from the apple        -   e. The apple should also not be floating    -   6. Add 100 mM CaCl₂ (in autoclaved dH₂O) and leave overnight        (room temperature)    -   7. Remove the CaCl₂ solution (room temperature)    -   8. Set the desired samples opposite to Sterile micro centrifuge        tubes    -   9. Remove the last water wash from the micro centrifuge tubes        and replace with 70% ethanol.    -   10. Leave in 70% ethanol for 30 mins-1 hour    -   11. Remove the 70% ethanol    -   12. Continue washing the apple slice with water with the same        technique as previously mentioned.        -   a. Make sure you change pasteur pipettes    -   14. Continue washing until the apple slices stop floating (at        least 4 times) with PBS    -   15. Remove the PBS and replace with 1% P/S PBS    -   16. Implant into animal model.

Example 2—Mouse Implantation

In vivo mouse implantation studies were performed to study in vivoeffects of scaffold biomaterial embodiments as described herein.

Results indicate that, following subcutaneous implantation in a mousemodel, full cell infiltration was observed (See FIG. 7; 1, 4 and 8 weeksafter implantation), with collagen deposition (FIG. 4A) and,importantly, angiogenesis with functional blood vessel formation within4 weeks post-implantation (FIGS. 4B and 8). When scaffolds wereimplanted in vivo, the minimum footprint promoted cell infiltration,angiogenesis and tissue repair and only a minimal inflammatory response(mainly produced by the surgery itself rather than the scaffold).Plant/fungus derived scaffolds were fully biocompatible in vivo in thesestudies. These scaffolds were also fully compatible with in vitrostudies as shown in (FIG. 5).

A Non-Biodegradable Biomaterial:

The field has been primarily focused on biodegradable materials;however, there are many issues with this approach in practice. Unlikemany commercial biomaterials, in certain embodiments the presentbiomaterials may be considered non-resorbable (i.e. may not fullybreakdown and be absorbed by the body) (see FIG. 9).

The non-resorbable characteristic of such scaffolds may offer certainadvantages over competing commercial products. By way of example, theymay be (i) more resistant to shape change and/or may hold their intendedgeometry over long periods of time; (ii) they may have a minimalfootprint compared to competing products, making them nearly invisibleto the body, eliciting almost no immune response; (iii) they may avoidthe production of by-products compared to resorbable materials, abreakdown of which may create an adverse immune response; and/or (iv)when resorbable biomaterials break down, the new regenerated tissues maybe damaged and may then be also eliminated; biomaterials as describedherein may, in certain examples, avoid such a situation.

In Vitro Study:

In vitro experiments described herein were carried out to confirm cellinvasion and proliferation inside the cellulose scaffold. Full cellinfiltration took many weeks when the first protocol (described inExample 1 above) was used. A modified protocol (also described inExample 1 above) was subsequently developed, which comprises theaddition of a calcium chloride wash (CaCl₂), which gave similar resultsbut within only one week (see FIG. 9).

In Vivo Study:

A preclinical trial was carried out on a mouse model to study theresponse to the subcutaneous implantation of 5×5×1 mm scaffolds over aperiod of 1, 4 and 8 weeks. Cellulose-based scaffolds originated fromapple, fennel, and asparagus, and chitin-based scaffolds originated fromwhite mushroom (see FIG. 6).

All scaffolds presented similar biocompatibility, with no rejection andthe observation of cell invasion and angiogenesis (formation of bloodvessels) in these studies.

Example 3—In Vivo Biocompatibility of Scaffold Biomaterials

To address the question of in vivo biocompatibility of the scaffoldbiomaterials, the response of the body to apple-derived cellulosescaffolds has been characterized. Macroscopic (˜25 mm³) cell-freecellulose biomaterials were produced and subcutaneously implanted in amouse model for 1, 4 and 8 weeks. Here, the immunological response ofimmunocompetent mice, deposition of extracellular matrix on thescaffolds and evidence of angiogenesis (vascularization) in theimplanted cellulose biomaterials was assessed. Notably, although aforeign body response was observed immediately post-implantation, asexpected for a surgical procedure, only a low immunological response wasobserved with no fatalities or noticeable infections whatsoever in allanimal groups by the completion of the study. Surrounding cells werealso found to invade the scaffold, mainly activated fibroblasts, anddeposit a new extracellular matrix. As well, the scaffold itself wasable to retain much of its original shape and structure over the 8-weekstudy. Importantly, the scaffolds clearly had a pro-angiogenic effect,resulting in the growth of functional blood vessels throughout theimplanted biomaterial. Taken together, this work demonstrates that thereis an relatively easy way to produce 3D cellulose scaffolds that arebiocompatible, becoming vascularized and integrated into surroundinghealthy tissues.

In these studies, the native hypanthium tissue of apples and aconvenient preparation methodology to create implantable cellulosescaffolds was used. To examine biocompatibility, scaffolds weresubcutaneously implanted in wild-type, immunocompetent mice (males andfemales; 6-9 weeks old). Following the implantation, the scaffolds wereresected at 1, 4 and 8 weeks and processed for histological analysis(H&E, Masson's Trichrome, anti-CD31 and anti-CD45 antibodies).Histological analysis revealed a characteristic foreign body response tothe scaffold 1 week post-implantation. However, the immune response wasobserved to gradually disappear by 8 weeks post-implantation. By 8weeks, there was no immune response in the surrounding dermis tissue,and there was active fibroblast migration within the cellulose scaffold.This was concomitant with the deposition of a new collagen extracellularmatrix. Furthermore, active blood vessel formation within the scaffoldwas observed throughout the period of study, indicating thepro-angiogenic properties of the native scaffolds. Finally, while thescaffolds retain much of their original shape, they do undergo a slowdeformation over the 8-week length of the study. Taken together, theseresults indicate that native cellulose scaffolds are biocompatible andmay exhibit potential as a surgical biomaterial.

Material and Methods

Animals All experimental procedures were approved by the Animal Care andUse Committee of the University of Ottawa. Wild-type C57BL/10ScSnJ mice(males and females; 6-9 weeks old; n=7 mice for each group) werepurchased from The Jackson Laboratory (Bar Harbor, Me., USA) and breedin our facilities. All animals were kept at constant room temperature(±22° C.) and humidity (˜52%). They were fed a normal chow diet and werekept under a controlled 12 hours light/dark cycle.

Cellulose scaffold preparation As described previously [27], McIntoshRed apples (Canada Fancy) were stored at 4° C. in the dark for a maximumof two weeks. In order to prepare apple sections, the fruit was cut witha mandolin slicer to a uniform thickness of 1.14±0.08 mm, measured witha Vernier caliper. Only the outer (hypanthium) tissue of the apple wasused. Slices containing visible ovary-core tissue were not used. Theslices were then cut parallel to the direction of the apple pedicel intosquare segments of 5.14±0.21 mm in length and with an area of 26.14±1.76mm². Apple tissue was then decellularized by using a protocol relatingto that of reference [14] for removing cellular material and DNA fromtissue samples while leaving behind an intact and three-dimensionalscaffold. Individual apple tissue samples were placed in sterilized 2.5ml microcentrifuge tubes and 2 ml of 0.1% sodium dodecyl sulphate (SDS;Sigma-Aldrich) solution was added to each tube. Samples were shaken for48 hours at 180 RPM at room temperature. The resultant cellulosescaffolds were then transferred into new sterile microcentrifuge tubes,washed and incubated for 12 hours in PBS (Sigma-Aldrich). To sterilizethe cellulose scaffold, they were incubated in 70% ethanol for 1 hourand then washed 12 times with PBS. The samples were then maintained inPBS with 1% streptomycin/penicillin (HyClone) and 1% amphotericin B(Wisent, QC, Canada). At this point, the samples were immediately usedor stored at 4° C. for no more than 2 weeks.

Cellulose implantation The mice were anesthetized using 2% IsofluraneUSP-PPC (Pharmaceutical partners of Canada, Richmond, ON, Canada) andtheir eyes protected by the application of ophthalmic liquid gel (AlcoCanada In., ON, Canada). To prepare the surgery sites, mouse back hairswere shaved and the skins were cleaned and sterilized using ENDURE 400Scrub-Stat4 Surgical Scrub (chlorhexidine gluconate, 4% solution; EcolabInc., Minnesota, USA) and Soluprep (2% w/v chlorhexidine and 70% v/visopropyl alcohol; 3M Canada, London, ON, Canada). To maintained animalhydration, 1 ml of 0.9% sodium chloride solution was administratedsubcutaneously (s.c.) (Hospira, Montreal, QC, Canada). During thesurgical procedures, we applied all sterility measures requested forsurvival surgeries. To implant the scaffolds, two 8 mm incisions weremade on the dorsal section of each mouse (upper and lower). Twocellulose scaffold samples were separately and independently implantedon each mouse. The incisions were then sutured using Surgipro IImonofilament polypropylene 6-0 (Covidien, Mass., USA) and transdermalbupivicaine 2% (as monohydrate; Chiron Compounding Pharmacy Inc.,Guelph, ON, Canada) was topically applied on surgery sites to preventinfection. Also, buprenorphine (as HCL) (0.03 mg/ml; Chiron CompoundingPharmacy Inc. Guelph, ON, Canada) was administrated s.c. as a painreliever. All animals were then carefully monitored for the next 3 daysby animal care services and received repetitions of the samepharmacological treatments.

Scaffold resections At 1, 4 and 8 weeks after scaffold implantation, themice were euthanized using CO₂ inhalation. After blood collection, thedorsal skin was carefully resected and immediately immersed in PBSsolution. The skin sections containing cellulose scaffolds were thenphotographed, cut and fixed in 10% formalin for at least 48 hours. Thesamples were then kept in 70% ethanol before being embedded in paraffinby the PALM Histology Core Facility of the University of Ottawa.

Histological analysis Serial 5 μm thick sections were cut, beginning at1 mm inside the cellulose scaffold, and stained with hematoxylin andeosin (H&E) and Masson's trichrome. For immunocytochemistry, heatinduced epitope retrieval was performed at 110° C. for 12 min withcitrate buffer (pH 6.0). Anti-CD31/PECAM1 (1:100; Novus Biologicals,NB100-2284, Oakville, ON, Canada), anti-alpha smooth muscle actin(1:1000, ab5694, abcam, Toronto, ON, Canada) and anti-CD45 (1:3000;ab10558, abcam, Toronto, ON, Canada) primary antibodies were incubatedfor an hour at room temperature. Blocking reagent (Background Sniper,Biocare, Medical, Concorde, Calif., USA) and detection system MACH 4(Biocare Medical, Concorde, Calif., USA) were applied according tocompany specifications. For the evaluation of cell infiltration,extracellular matrix deposition and vascularisation (angiogenesis),micrographs were captured using Zeiss MIRAX MIDI Slide Scanner (Zeiss,Toronto, Canada) equipped with 40× objective and analysed usingPannoramic Viewer (3DHISTECH Ltd., Budapest, Hungary) and ImageJsoftware. The scoring of inflammation was evaluated by a pathologist.The scoring was subjectively assigned by qualitative analysis of themagnitude of the total foreign response as well, the cell populationproportions within the foreign response.

Scanning electron microscopy (SEM) The structure of cellulose wasstudied using a scanning electron microscopy. Globally, scaffolds weredehydrated through successive gradients of ethanol (50%, 70%, 95% and100%). Samples were then gold-coated at a current of 15 mA for 3 minuteswith a Hitachi E-1010 ion sputter device. SEM imaging was conducted atvoltages ranging from 2.00-10.0 kV on a JSM-7500F Field Emission SEM(JEOL, Peabody, Mass., USA).

Statistical analysis All values reported here are the average±standarddeviations. Statistical analyses were performed with one-way ANOVA byusing SigmaStat 3.5 software (Dundas Software Ltd, Germany). A value ofp<0.05 was considered statistically significant.

Results

Scaffold Preparation Cellulose scaffolds were prepared from apple tissueusing a decellularization technique relating to that previouslydescribed [27]. All scaffolds were cut to a size of5.14±0.21×5.14±0.21×1.14±0.08 mm (FIG. 11A), decellularized and preparedfor implantation (FIG. 11B). The scaffolds appear translucent afterdecellularization due to the loss of all plant cellular material anddebris. The removal of apple cells was also confirmed with histologicalobservation (FIG. 11C) and scanning electron microscopy (FIG. 11D).Analysis of the histological images and the measurement of the averagewall thickness (4.04±1.4 μm) reveal that under the experimentalconditions the cellulose scaffolds were highly porous, capable of beinginvaded by nearby cells and results in an acellular cellulose scaffoldthat maintains its shape.

Implantation of Cellulose Scaffolds Two independent skin incisions (8mm) were produced on the back of each mouse to create small pouches forthe biomaterial implantation (FIG. 12A). One cellulose scaffold (FIG.12B) was implanted in each subcutaneous pouch. Throughout the study,there were no cases of mice exhibiting any pain behaviour that may havebeen induced by the cellulose scaffold implantation and none of themhave displaying any symptoms of visible inflammation or infection. Thecellulose scaffolds were resected at 1 week, 4 weeks and 8 weeks aftertheir implantation and were photographed to measure the change inscaffold dimensions (FIGS. 12D-F). At all-time points, healthy tissuecan be observed surrounding the cellulose scaffold with the presence ofblood vessels, that are proximal or in direct contact, and the scaffoldsretain their square shape. The pre-implantation scaffold had an area of26.3±1.98 mm² and it was observed to slowly decrease as function oftheir implantation time base on the scaffold area that is visible to thenaked eye on the skin (FIG. 12G). At 8 weeks post-implantation, thescaffold dimensions reach a near plateau measurement of 13.82±3.88 mm²demonstrating an approximate 12 mm² (48%) change over the course of thisstudy.

Biocompatibility and cell infiltration in plant derived cellulosescaffolds Scaffold biocompatibility and cell infiltration was examinedwith H&E staining of fixed cellulose scaffolds at 1, 4 and 8 weeksfollowing their implantation (FIG. 13). The global views of longitudinalsection of representative cellulose scaffolds are shown in FIGS. 13A-C.The scaffolds are implanted under the muscular layer of the dermis.Interstitial fluids, stained in pink, can be seen throughout theimplanted scaffold, in contrast to a non-implanted scaffold (see FIG.11C), highlighting their high porosity and permeability. Within theglobal view it was observed that the scaffold maintains its generalshape throughout the study. In FIGS. 13D-F, a magnified section of theperimeter of the scaffold is shown at each post-implantation timepoints. At 1 week, the dermis tissue surrounding implant displayssymptoms of an acute moderate to severe immune response (qualitativestudy performed by a pathologist) (FIG. 13D). As well a dense layer ofcells can be seen infiltrating into the cellulose scaffolds. Thepopulation of cells within the scaffold at 1 week consist mainly ofgranulocytes, specifically; polymorphonuclear (PMN) and eosinophils(FIG. 13D). There is also a population of dead cells and apparent celldebris. Importantly, all of these observations are completely consistentwith an expected acute foreign body reaction that follows implantation[84-86]. At the 4 week point, a stark difference in both the surroundingepidermis tissue and in the cell population migrating into the cellulosescaffold was observed (FIG. 13E). The epidermis tissue surrounding thecellulose scaffold has a decreased immune response, now scored as mildto low. The population of cells within the epidermis surroundingscaffolds now contain higher levels of macrophages and lymphocytes (FIG.13E). This is an anticipated characteristic of the foreign body reactionto an implanted biomaterial, demonstrating the scaffold cleaning process[84-86]. There is also an increase in the population of multinucleatedcells within the interior of the scaffold as part of an inflammatoryresponse (FIG. 13E). Finally, 8 weeks post-implantation, the immuneresponse apparent at 1 and 4 weeks has completely disappeared (FIG.13F), with the epidermis tissue now appearing normal. In fact, theepidermis tissue in contact with the cellulose scaffold contains thesame structures as normal epidermis tissue. In the cellulose scaffoldperimeter there is now a lower density of cells due to the decreasedinflammation and notably, there are no fragmented dead cells present.Instead, the population of cells now contain an elevated level ofmacrophages, multinucleated cells and active fibroblasts. The activefibroblasts (appearing spindle shaped), can be observed migrating fromthe surrounding epidermis into the cellulose scaffold. In fact,fibroblasts were found throughout the cellulose scaffold. These resultsdemonstrate that by 8 weeks post-implantation, the cellulose scaffoldhas been accepted by the host. In parallel with the H&E inflammationanalysis, anti-CD45 staining was performed to evaluate the level ofinflammation throughout the scaffold and surrounding dermis tissue(FIGS. 3G-I). It is clear that the inflammation throughout the dermisand within the scaffold is elevated after 1 week. However, the amount ofleukocytes significantly decreases in the surrounding dermis andscaffold over the implantation time reaching a near basal level at 8weeks.

Extracellular Matrix Deposition in the Cellulose Scaffolds The presenceof active fibroblasts led us to question if the cellulose scaffold wasacting as a substrate for the deposition of new extracellular matrix.This was determined using Masson's Trichrome staining of fixed cellulosescaffolds slides at each time point following implantation (FIG. 14). At1 week post-implantation, the histological study shows the absence ofcollagen structures inside the collagen scaffold (FIGS. 14A, D, and G).As fibroblast cells invade the scaffold, as seen with H&E staining andconfirmed by anti-alpha smooth muscle actin staining (data not shown),collagen deposits inside the cellulose scaffold can be observed after 4weeks (FIGS. 14B, E, and H). At 8 weeks (FIGS. 14C, F and I) thecollagen network is clearly visible inside the cavities of the cellulosescaffold. The complexity of the deposited collagen network ishighlighted in FIG. 14I, where we can detect individual collagen fiberswithin the collagen matrix. This is in contrast to the characteristichigh density, thick, cable-like organization of collagen found in scartissue.

Vascularization of the Cellulose Scaffolds Capillaries ranging from 8 to25 μm in diameter were also identified within the scaffolds as early as1 week post-implantation. At 4 week and 8 week post implantation, bloodvessels and capillaries can be observed extensively within the scaffoldand the surrounding dermal tissue. We observed blood vessels presence onthe cellulose scaffold and in surrounding dermis in the macroscopicphotos taken during the resection (FIG. 15A). Multiple cross sections ofblood vessels, with the presence of red blood cells (RBCs), areidentified within 4 weeks of scaffold implantations (FIG. 15B; H&Estain). The same results are obtained 8 weeks after implantation wherecapillaries with RBC and endothelial cells are clearly seen (FIG. 15C;Masson's Trichrome). All results on blood vessels formation were alsoconfirmed with anti-CD31 staining to identify endothelial cells in thescaffold (FIG. 15D).

Analysis

In this study, the in vivo biocompatibility of acellular cellulosescaffolds derived from apple hypanthium tissue was assessed. To thisend, acellular cellulose scaffolds were subcutaneously implanted withinimmunocompetent mice to establish their biocompatibility. The datareveals that the implanted scaffolds demonstrate a low inflammatoryresponse, promote cell invasion and extracellular matrix deposition, andact as a pro-angiogenic environment. Remarkably, none of the mice inthis study died or demonstrated any symptoms of implant rejection suchas edema, exudates or discomfort during the course of this researchindicative of a successful implantation of the cellulose scaffolds.These implanted scaffolds are composed of a porous network of cavitiesin which the original host plant cells resided [69]. This architectureefficiently facilitates transfer of nutrients throughout the planttissue. As shown here and in a previous study, apple tissues may bedecellularized [27]. This simple treatment changes the appearance of thehypanthium tissue so that it becomes transparent, as a result of theremoval of cellular materials.

After implantation, the results indicate that the scaffolds are rapidlyinfiltrated with host cells, which begin with inflammatory cells.Consistent with previous findings, the immune response of the hostanimals followed a well-known timeline [84-88], ultimately demonstratingbiocompatibility. As expected, the cell population within the scaffoldafter 1 week post-implantation are mainly granulocytes, specifically;polymorphonuclear (PMN) and eosinophils, constituting a clearinflammatory response. The production of a provisional matrix around thescaffold was also observed resulting in an inflamed appearance in thetissue surrounding the scaffold [84-88]. This is not unexpected and isthe result of the foreign material as well as a response to the surgicalprocedure [84-88]. Four weeks post implantation, the population of cellswithin the scaffold have evolved and are now lymphocytes, monocytes,macrophages, foreign body multinucleated cells as well as scatteredeosinophils. Typical with chronic inflammation, the cellular debrispresent in the provisional matrix at 1 week, is now being cleared by thehost immune system [84-88]. At 8 weeks, the cellulose scaffold is nowvoid of all provisional matrix and cellular debris and low levels ofmacrophages and foreign body multinucleated cells are still visiblewithin the scaffold. Consistent with the immune response within thecellulose scaffold, the surrounding tissue is observed to return to itsoriginal physiology. In fact, at 8 weeks post-implantation, thesurrounding tissue was nearly similar to control tissue. Although theimmune response and inflammation at 8 weeks is low, low levels ofmacrophages can be observed within the scaffold. Although traditionallyassociated with inflammation, macrophages have beneficial rolesconsistent with our findings. Specifically, macrophages are also knownto secrete growth and pro-angiogenic factors, ECM proteins andpro-fibrogenic factors that actively regulate the fibro-proliferationand angiogenesis in tissue repair and regeneration [86]. Regardless, thevast population of cells within the scaffold after 8 weeks are nowreactive fibroblasts. These cells are altering the microenvironment ofthe scaffold through the secretion of a new collagen extracellularmatrix. The new matrix displayed a remarkably low density compared,suggestive of regeneration as opposed to the characteristic highdensity, cable-like organization of collagen found in scar tissues [89].

These data also demonstrate that the scaffolds are pro-angiogenic, whichmay facilitate blood transport from the surrounding tissue [90]. As withnative tissue, limited blood supply to the scaffold may result inischemia and potentially necrosis. Interestingly, it was demonstratedthat bioceramics with pore diameters lower than 400 μm resulted in adecrease in the growth of blood vessels and limits the size of bloodvessel diameter in in vivo implantations. The porous structure of thecell wall architecture is composed of overlapping cell wall cavitieswith diameters ranging from 100-300 μm with manual interconnectiondistance of 4.04±1.4 μm. As such, the high porosity size and lowvolume-fraction of the cellulose scaffolds are consistent with thepromotion of blood vessel formation. Taken together, the cellulosescaffold now appears to be void of the provisional matrix and fullyaccepted as a subcutaneous implant.

We also observed a decrease in the scaffold area over time, but it doesnot appear that the cellulose scaffold is in the processes ofdegradation. Rather, the change in area appears to be due to thecollapse of the cell wall cavities on the perimeter of the scaffoldresulting from the active movement of the mouse. Active biologicaldegradation is not expected to be possible as mammals lack theappropriate enzymes to digest plant-synthesized cellulose [91,92].Moreover, the highly crystalline form of cellulose that is found inplant tissues is also known to be resistant to degradation in mammals[92]. Alternatively, it has been demonstrated that in vivo celluloseimplants can be chemically activated in order to be more easily degraded[93]. However, highly crystalline forms of cellulose have some of thelowest reported immunological responses [92].

A large variety of clinically approved biomaterials are used to treatspecific conditions within patients [1]. Such biomaterials may bederived from human and animal tissues, synthetic polymers, as well asmaterials such as titanium and ceramics[1,2,26,49,50,53,54,56,74,76,94-106]. However, these approaches are notwithout disadvantages that arise from concerns about the source,production costs and/or widespread availability [48]. There is currentlyan intense interest in developing resorbable biomaterials that willdegrade in vivo and only act as a temporary scaffold that will promoteand support the repair or regeneration of damaged/diseased tissue [49].Although this is an appealing scenario, newly formed structures are alsofound to collapse as the scaffold degrade [53,64,107-109]. Moreover, theproducts of degradation can also be found to have toxic or undesirableside-effects [53,110,111]. For example, the reconstruction of the earhas become a well-known challenge in tissue engineering. Early studieshave employed scaffolds in the shape of an ear that are produced fromanimal or human derived cartilage [53,58,59,61,63,64]. However, afterimplantation and eventual scaffold degradation, the ear is often foundto collapse or deform [60-62]. Recent strategies have now opted tocreate biological composite materials composed of both a titanium frameembedded in a biological matrix [53].

Results provided herein suggest that plant-derived cellulosebiomaterials may offer one potential approach for the production ofimplantable scaffolds. This approach may be complementary to bacterialcellulose strategies [66,69-71,73,80,83,102,106,112-115]. However,results provided herein suggest that plant derived materials may be costeffective to produce, may be convenient to prepare for implantation, mayexhibit clear biocompatibility, may feature an ability to retain shapewhile supporting the production of natural host extracellular matrix,and/or may promote vascularization. In previous work, the inventors haveshown that scaffolds may be functionalized with proteins prior toculture in vitro. It is contemplated herein that the use of scaffoldsurface functionalization with growth factors and matrix proteins, forexample, may be used to promote the invasion of specific cell types,further minimize the early immune response, and/or to promotevascularization. Moreover, cellulose scaffolds may easily be formed intospecific shapes and sizes, offering an opportunity to create new tissueswith specific geometrical properties. As shown herein, acellularcellulose scaffolds are biocompatible in vivo in immunocompetent miceunder the conditions tested, and may be considered as a new strategyfor, for example, tissue regeneration.

Example 4—Additional Decellularisation Protocol Example

An additional decellularlisation protocol is described herein. In thisexample, plants were chilled in a −20° C. freezer for a duration of 5minutes to allow the soft tissue to firm up. A mandolin slicer wasutilized to section the chilled plant tissue to a uniform thicknessmeasured with a vernier caliper. The slices were then cut into segmentsand then decellularized by using a modified mammalian tissue protocolfor removing cellular material and DNA from tissue samples while leavingbehind an intact and three-dimensional scaffold. The protocol wasmodified from a protocol for mammalian tissue (Ott et al., 2008).Individual tissue samples were placed in sterilized 2.5 mLmicrocentrifuge tubes and 2 mL of 0.5% sodium dodecyl sulphate (SDS;Sigma-Aldrich) solution was added to each tube. Samples were shaken for12 hours at 160 RPM at room temperature. The resultant cellulosescaffolds were then transferred into new sterile microcentrifuge tubes,washed and incubated for 6 hours in PBS (Sigma-Aldrich) with 1%streptomycin/penicillin (HyClone) and 1% amphotericin B (Wisent). Atthis point, the samples were immediately used or stored in PBS at 4° C.for no more than 2 weeks. The resultant decellularized cellulosescaffolds can be observed in FIGS. 1 A and B.

Example 5—Two Dimensional (2D) and Three Dimensional (3D) Cell CultureIn Vitro—Scaffold Implantation, Cell Adhesion, and Cell Proliferation

C2C12 mouse myoblasts, NIH3T3 mouse fibroblasts and HeLa humanepithelial cell lines were used in this study (all obtained from theAmerican Type Culture Collection (ATCC)). The cells were selected asthey represent the most common cell type used in cell biologylaboratories. 2D conventional cell culture was employed to harvest theabove-mentioned cells for the scaffold implantation. Cells were culturedin standard cell culture media (high glucose DMEM (HyClone)),supplemented with 10% fetal bovine serum (HyClone), 1%penicillin/streptomycin (HyClone) and 1% amphotericin B (Wisent) at 37°C. and 5% CO₂ in T75 flasks (Thermo Scientific). Culture media wasexchanged every second day and the cells were passaged at 80%confluence.

The scaffold seeding procedure took place in 24-well tissue cultureplates. The wells were individually coated with polydimethylisiloxane(PDMS) to create a hydrophobic surface in order to make the cellulosescaffold the only adherable surface. A 1:10 solution of curing agent:elastomer (Sylgard 184, Ellsworth Adhesives) was coated into each wellsurface. The PDMS was allowed to be cure for 2 hours at 80° C. ThePDMS-24 well plates were allowed to cool to room temperature and thenrinsed with sterile PBS. Scaffolds were cut into 0.5×0.5 cm pieces andplaced within each well. The C2C12, NIH3T3 and HeLa were adhered andaliquoted to their correct concentration. A 40 μL droplet containing6×10⁶ cells were carefully formed on top of each scaffold. The sampleswere placed in the incubator for 6 hours to allow the cells to adhere tothe scaffolds. Subsequently, 2 mL of DMEM was added to each well and thesamples were incubated for 48 hours. At this point, samples containingmammalian cells were then carefully transferred into new 24-wellPDMS-coated tissue culture plates. For continued cell proliferation, theculture media was exchanged every day and scaffolds were moved into new24-well plates every 2 weeks.

The adhesion and proliferation of the mammalian cells were monitored anddetermined using immunofluorescent microscopy. FIGS. 5A-C, 16 and 17demonstrate the adhesion and continuous proliferation of the cell linesused.

Example 6—Salt Pretreatment Effects, and Scaffold BiomaterialFunctionalization

Decellularization was used to obtain the 3D cellulose scaffold void ofnative cells and nucleic acids. The surfactant sodium dodecyl sulfate(SDS) was used to accomplish the decellularization. The SDS was removedbefore the scaffold is repopulated with new cells; since the cells willotherwise perish. With small scaffolds, the concentration of SDS may below; however, for larger objects a higher concentration of SDS may beused to undergo complete decellularization. Remnant SDS may be removedby sufficient washing, particularly when low concentrations of SDS areused. Higher concentrations of SDS may become difficult and timeconsuming to remove via washing alone in certain cases. As describedherein, the addition of CaCl₂ may allow for the easy removal of residualSDS from the decellularized scaffold. Without wishing to be bound bytheory, the principle behind this concept is believed to use the saltbuffer to force the SDS into micelles. A sufficiently high saltconcentration may be used to stimulate adequate micelle formation, and asalt concentration which is too high may cause the salt to crash outonto the biomaterial. The salt residue may be removed by severaltechniques, such as incubating with dH₂O, acetic acid, or DMSO.Sonication may also be used to remove tightly bound debris. Theconcentration of CaCl₂ may be dependent on the amount of residual SDS.In this study, decellularization was accomplished by using 0.1% SDS inwater. The concentration of CaCl₂ may depend on the amount of SDS usedfor decellularization, as shown in FIG. 18. At a concentration of 100mM, a moderate amount of salt/micelles crashed out onto the scaffold(FIG. 19A). The salt residue was effectively removed by incubating thescaffold in dH₂O (FIG. 19B).

Improved cell growth was obtained after the removal of the residual SDSand salt (FIG. 20). The addition of the salt may allow for the easyremoval of the residual SDS; however, salt that crashes out onto thebiomaterial should also be removed to avoid tonicity issues. After thesalt forces the SDS into micelles, the next step is to remove the salt.The salt residue may be removed with various techniques such assonication treatment, water incubation, acetic acid incubation, and DMSOincubation (FIG. 20).

In addition to CaCl₂, other salts may also be used remove the residualSDS from the biomaterial (FIG. 21). Washing the biomaterials with a saltthat has a divalent cation led to greater cell growth than theirmonovalent counterparts, likely because the divalent cations promotedtighter SDS micelle formation (FIG. 21).

In certain embodiments, the addition of the salt may alter the criticalmicelle concentration (CMC) of the surfactant. At a certainconcentration known as the cloud point, a phase transition may occur,and the micelles become insoluble and may be readily washed away.

Different salt compounds may be used to accomplish the task of removingthe residual SDS from the biomaterial. PBS, KCl, CaCl₂, MgCl₂, CuSO₄,KH₂PO₄, MgSO₄, Na₂CO₃, and sodium ibuprofenate (all 100 mM) were used asa salt wash to clean the biomaterial, and remove residual SDS. Each salttreatment shown in FIG. 21 allowed for cell growth; however, the saltswith divalent cations (CaCl₂ and MgCl₂) as well as the carbonate aniongroup promoted greater cell growth.

Biomaterial Functionalization

The cellulose structure may be biochemically functionalized depending onthe intended use of the biomaterial. As will be understood, suchmodification may expand potential uses and applications. Cellulose, forexample, has free hydroxyl groups which may be exploited to conjugatethe material with different molecules.

Two commonly used classes of reactions for this type of modification areacylation and alkylation reactions. These reactions may allow forhydrocarbon chains of various lengths to be attached to the cellulosestructure via the free hydroxyl groups. The different chain lengths andshapes may be useful when steric hindrance is a factor, for example. Theuse of larger chains may decrease the steric hindrance, and vice-versa.Acylation reactions using dicarboxylic acids may provide possibilitiesto functionalize the biomaterial. Some classes of dicarboxylic acidsthat may be used may include, but are not limited to, linear saturateddicarboxylic acids, branched dicarboxylic acids, unsaturateddicarboxylic acids, substituted dicarboxylic acids, and aromaticdicarboxylic acids. In addition to acylation and alkylation reactions,other compounds may be used to mediate the connection between thefunctional group and the cellulose such as compounds containing boron,sulfur, nitrogen, and/or phosphorous, for example.

Different functional groups may be added to the other end of the chainin order to fulfill a certain function. These functional groups mayinclude, but are not limited to, groups containing hydrocarbons, oxygen,nitrogen, sulfur, phosphorous, boron, and/or halogens. The choice offunctional group may depend on the intended application. For example, ifthe intended application is to prevent cell growth in certain areas, asteric non-polar hydrocarbon functional group may be used; conversely,if the intended application is to promote cell growth, a carboxylic acidmay be chosen, so that extracellular matrix proteins, such as collagen,may bind to the cellulose.

Different elements of the cell wall may allow for enhancing certainstructural properties of the biomaterial. The secondary cell wallstructures of asparagus and apple tissue may contain, for example,pectin and lignin (FIG. 22) to lend strength to the biomaterial.

As will be understood, the scaffold biomaterials are not limited tocellulose. Many other cell wall structures may be used for thebiomaterial. In FIG. 22, there are also cinnamaldehydes, pectin, andlignin, in addition to the cellulose shown. These additional secondarycell wall structures may also be functionalized.

Chemical modification of the cellulose may allow for control over thechemical and physical properties of the biomaterial. As a result, thebiomaterial may be specialized for specific purposes. For example,patterned cell growth may be achieved by inhibiting cell growth incertain areas (temporarily or permanently) and promoting it in others.Moreover cell type specific molecules may be introduced to thebiomaterial through these functionalization methods to promote thegrowth/invasion/differentiation of specific cells types. Thefunctionalization of the biomaterial may also allow for the recreationof biologically relevant microenvironments, which are involved in propercell function and tissue engineering.

Example 7—Surface Biomodification

Native cellulose can support mammalian cells, including C2C12 myoblast,3T3 fibroblast and human epithelial HeLa cells. However, a functionalbiomaterial may further able to be chemically and mechanically tuned tosuit the particular intended use. Two different techniques were used inthese experiments to change the stiffness of the decellularizedcellulose scaffold. Additionally, phase contrast images demonstrate thatthe biomaterials still support mammalian cell culture after chemical andphysical modification.

In order to functionalize scaffolds with collagen, samples wereincubated for 6 hours in a solution of 10% acetic acid and 1 mg/mL rattail collagen type I (Invitrogen), followed by washing in PBS beforeuse. To chemically cross-link the scaffolds, the samples were incubatedin a 1% EM-grade glutaraldehyde solution (Sigma-Aldrich) for 6 hours.The scaffolds were then rinsed in PBS and incubated in a solution of 1%sodium borohydride (Sigma-Aldrich) overnight in order to reduce anyunreacted glutaraldehyde. Prior to seeding cells into the scaffolds, allsamples (native, collagen coated, or cross-linked) were incubated inmammalian cell culture medium (described below) for 12 hours in astandard tissue culture incubator maintained at 37° C. with 5% CO₂.Results are shown in FIG. 23A-D. The native tissue and unmodifiedscaffolds do not display any significant difference in mechanicalproperties. Both the collagen functionalized and chemically cross-linkedscaffolds displayed a significant increase in elasticity compared to theDMEM scaffolds. The decellularized (SDS), collagen functionalized(SDS+Coll) and glutaraldheyde cross-linked (SDS+GA) scaffolds allsupported the growth of C2C12 cells under the experimental conditions.

Example 8—Cellulose Scaffolds and Moulding Techniques, Coatings

We have previously shown how cellulose scaffolds may act as standalone3D biomaterials. Here we show how decellularized cellulose may be cutinto different macroscopic shapes (FIG. 24: rings). C2C12 mouse myoblastcells were seeded onto the biomaterial, and the cells were allowed toproliferate and invade the scaffold for two weeks. After two weeks, thestructures were full of cells (FIG. 24). The biomaterials may be used incombination with conventional moulding techniques as well. In thisstudy, we show how a cellulose construct may be used for both temporaryand permanent inverse moulding using gelatin and collagen respectively(FIG. 24B-C). Gelatin has a melting temperature of 32° C. For thetemporary inverse mould, the cells were resuspended in a 10% gelatinsolution in cell culture media at 37° C. Shortly after the cells wereseeded onto the biomaterial, the gelatin solution cooled below itsmelting temperature and solidified. The formation of the gelatin gelgave the cells time to attach to the substrate. Once the gelatin gel washeated to 37° C. after being placed in the incubator, the gelatin meltedaway while the cells remained on the biomaterial. Conversely, thecellulose may also act as an inverse mould for permanent gels when thegel is desired. For the permanent inverse mould, the cellulose wascovered in a collagen solution containing cells (FIG. 24C). The collagensolution rapidly solidified and formed a permanent gel containing thebiomaterial and the cells.

The moulding techniques may further apply to other hydrogels, not simplygelatin and collagen. Other possible gels may include, but are notlimited to, for example, agarose, polyurethane, polyethylene glycol,xanthan, methyl cellulose, alginate, hyaluronan, carboxymethylcellulose,chitosan, polyacrylic acid, polyvinyl alcohol, polyester, hydrocolloids,gum arabic, pectin, and/or dextran. Hydrogels may be impregnated withother compounds as well, such as growth factors, drugs, etc. Such gelsmay also be functionalized with active side chains. As a result, it iscontemplated that, for example, the cellulose may have onefunctionality, and the hydrogel may have a second functionality.Moreover, multiple hydrogels with multiple functionalities may be usedin combination in certain embodiments. Finally, these gels may betemporary and melt away over time, and/or may be cross-linked to theoriginal cellulose or chitin scaffold to create multi-functionalmaterials with two or more mechanical/chemical properties that may betime-dependent or time-independent.

Additional elements/compounds may be used to coat the surface, or may bebound to the biomaterial through functionalization. The choice of theadditional element depends on intended application. For example, if thebiomaterial is for promoting nerve regeneration, Nerve Growth Factor(NGF) protein may be added. Conversely, if the biomaterial is for drugdelivery, a virus capsule containing the drug may be used. Moreover, thebiomaterial may be coated with, for example, an ibuprofen salt if animmune response is problematic. It is contemplated that various elementsmay be added to the biomaterial. These elements may include, but are notlimited to, proteins (e.g. collagen, elastin, and integrin), nucleicacids (e.g. DNA, RNA, and siRNA), fatty acids (e.g. stearic acid,palmitic acid, and linoleic acid), metabolites (e.g. aspartic acid,vitamin B2, and glycerol), ligands (e.g. vitamin D, testosterone, andinsulin), antigens (e.g. peptides, polysaccharides, and lipids),antibodies (e.g. IgA, IgE, and IgG), viruses (e.g. HIV, HEP C, andcowpox), synthetic polymers (e.g. nylon, polyester, and Teflon),functional groups (carboxylic acids, esters, and imides), drugs (e.g.hydrocodone, amoxicillin, Plavix, for example), vesicles (e.g. vacuoles,transport vesicles, and secretion vesicles), organic molecules (e.g.carbohydrates, ligases, and vitamins), and/or inorganic molecules (e.g.iron, titanium, and gold). In addition, bacteria (such as, but notlimited to bifidobacteria) may be added to alter/control the microbiome.Where cell specificity is desired, a cell recruiting factor may beincluded, for example.

Supporting Structures for the Biomaterial

Additional elements/compounds may be used as supporting structures tothe biomaterial. The choice of the additional element may depend on theintended application. For example, if the biomaterial is to sustain aconstant load or keep its shape, a titanium structure may be included.By way of example, such elements/components may include titanium, lowC-steel, aluminium, Co—Cr alloys, stainless steel type 316, PMMA cement,ultrahigh MW PE, etc. In certain embodiments, such elements may be addedwithin (inside) the biomaterial, outside the biomaterial, or both. Incertain embodiments, such elements/compounds may include those whichhave already passed FDA approval.

Example 9—Cell Invasion and Proliferation

Confocal laser scanning microscopy was used to image ˜300 μm z sectionsof the top and bottom of the cellulose constructs. Both sides wereimaged because the depth of field was less than the ˜1.2 mm thick ring.FIG. 25 shows the xy and zy projections of the cells on the cellulosebiomaterial. The nuclei of the cells (blue) were found along thecellulose cell walls (red) (FIG. 25 xy projections). Orthogonal views ofconfocal scans reveal that the cells invaded the scaffold (FIG. 25 zyprojections). The confocal imaging allowed for the cell invasion andproliferation to be quantified (FIG. 26). The cell nuclei images werethresholded using the ImageJ adaptive threshold plugin, and the analyzeparticles plugin was used to measure the total nuclear area. Initially,the cells were seeded onto the top of the sample. The ratio of thenuclear area covering the top and bottom of the biomaterial was used tomeasure the cell invasion. There were no statistical differences betweenthe three different conditions for the cell invasion (FIG. 26). In fact,the top:bottom ratio was close to 1 (FIG. 26). The total nuclear area ofthe imaged sections was calculated to compare the proliferation of thecells on each condition. It was found that the total nuclear area wasnot significantly different between the three conditions. As a result,temporary and permanent inverse moulding did not affect cellproliferation under the conditions tested.

Moulding techniques, as well as functionalization techniques, may beused to join together different structures. As a result, in certainembodiments, large complex structures may be created to mimic in vivotissues, for example.

Example 10—Artificially Fabricated Architecture within Plant-DerivedDecellularized Cellulose Scaffolds

Artificial fabrication of architecture within the plant cellulosescaffolds was performed to demonstrate the feasibility of creatingdifferent architecture for specific purposes such as increasing hostcell migration into the cellulose scaffold. Results are shown in FIG.27, where such artificial architecture was created in apple-derivedcellulose-based scaffolds.

In these studies, mice were anesthetized using 2% Isoflurane USP-PPC(Pharmaceutical partners of Canada, Richmond, ON, Canada) with the eyesprotected with the application of ophthalmic liquid gel (Alco CanadaIn., ON, Canada). The mouse back hairs were shaved with the underlyingskin cleaned and sterilized using ENDURE 400 Scrub-Stat4 Surgical Scrub(chlorhexidine gluconate, 4% solution; Ecolab Inc., Minnesota, USA) andSoluprep (2% w/v chlorhexidine and 70% v/v isopropyl alcohol; 3M Canada,London, ON, Canada). Animal hydration was maintained, via subcutaneousinjection (s.c) of 1 ml of 0.9% sodium chloride solution (Hospira,Montreal, QC, Canada). Throughout the surgical procedures all strictsterility measures were upheld for survival surgeries. To implant thescaffolds, two 8 mm incisions were cut on the dorsal section of eachmouse (upper and lower). Two cellulose scaffold samples were separatelyand independently implanted into each mouse. The incisions were thensutured using Surgipro II monofilament polypropylene 6-0 (Covidien,Mass., USA) and transdermal bupivicaine 2% (as monohydrate; ChironCompounding Pharmacy Inc., Guelph, ON, Canada) was topically applied tothe surgery sites to prevent infection. Additionally, buprenorphine (asHCL) (0.03 mg/ml; Chiron Compounding Pharmacy Inc. Guelph, ON, Canada)was administrated s. c. as a pain reliever. All animals were thencarefully monitored for the following 3 days by animal care services andreceived additional treatment of the same pharmacological treatments. At1 and 4 weeks after scaffold implantation, the mice were euthanizedusing CO₂ inhalation. The dorsal skin was carefully resected andimmediately immersed in PBS solution. The skin sections containingcellulose scaffolds were then photographed, cut and fixed in 10%formalin for at least 48 hours. The samples were then kept in 70%ethanol before being embedded in paraffin by the PALM Histology CoreFacility of the University of Ottawa.

Results are shown in FIG. 27. Two different architectures were createdwithin the decellularized cellulose scaffolds to demonstrate thefeasibility of creating different architectures with the biomaterial forspecific purposes such as increase the host cells migration into thecellulose scaffold. In FIG. 27A a 1 mm biopsy punch was used to createfive negative cylindrical spaces within the cellulose scaffold.Conversely, in FIG. 27B a 3 mm biopsy punch was used to create a singlecentered negative space. Only after 4 weeks implantation increased bloodvessel formation could be observed stemming directly from the artificialderived negative spaces (FIGS. 27C and D). In 28C blood vessels are ineach of four corners of the biomaterial suggesting the increase ofvascularization within the artificial derived negative space. Similarly,in 27D blood vessels can be observed on the top of the cellulosescaffold suggesting that the blood vessels travelled through thecellulose scaffold. Cross sections of representative cellulose scaffoldsstained with H&E (FIG. 27E-F).

Example 11—Various Examples of Cellulose-Based Origin Tissues andStructures in the Plant Kingdom

FIG. 28 provides various examples of cellulose-based origin tissue andstructures selected from the plant kingdom, shown at 4 weeks and/or 8weeks. This Figure shows pictures depicting cellulose scaffolds fromvarious sources, their resection and histology after 4 weeks and/or 8weeks, as indicated.

In these studies, various plant derived cellulose scaffolds weresubcutaneously implanted within mice to assess biocompatibility at 4weeks and/or 8 weeks. Selective tissue of various plants were implantedfor a period of 4 or 8 weeks to assess the biocompatibility of plantderived cellulose and the plant architecture on in vivo host cellmigration. In all examples, cell migration and proliferation into thecellulose scaffold was observed, highlighting the biocompatibility ofthe plant derived cellulose scaffolds in these experiments. Thesubcutaneous implantations of cellulose scaffold biomaterials wereperformed on the dorsal region of a C57BL/10ScSnJ mouse model by smallskin incisions (8 mm). Each implant was measured before theirimplantation for scaffold area comparison (first column: CelluloseScaffold). Cellulose grafts were resected (second column: Resection) at4 or 8 weeks as indicated. Serial 5 μm thick sections were cut,beginning at 1 mm inside the cellulose scaffold, and stained withhematoxylin-eosin (H&E) (third column: Histology). For the evaluation ofcell infiltration, micrographs were captured using Zeiss MIRAX MIDISlide Scanner (Zeiss, Toronto, Canada) equipped with 40× objective andanalysed using Pannoramic Viewer (3DHISTECH Ltd., Budapest, Hungary) andImageJ software.

Example 12—Biocompatibility of Subcutaneously Implanted Plant-DerivedCellulose Biomaterials (Prosthetic-Esthetic)

Building on Example 3 herein above, cellulose scaffold implantation andresection was performed to assess subcutaneous implants. Experimentalresults are shown in FIG. 29. The subcutaneous implantations ofcellulose scaffold biomaterials were performed on the dorsal region of aC57BL/10ScSnJ mouse model by small skin incisions (8 mm) (FIG. 29A).Each implant was measured before their implantation for scaffold areacomparison (FIG. 29B). Celluose scaffolds were resected at 1 week (FIG.29D), 4 weeks (FIG. 29E) and 8 weeks (FIG. 29F) after the surgeries andmacroscopic pictures were taken (control skin in FIG. 29C). At each timepoint, blood vessels are clearly integrated with the cellulose implantdemonstrating the biocompatibility. As well there is no detected acuteor chronic inflammation in the tissue surrounding the implant. Thechanges in cellulose scaffold surface area over time are presented inFIG. 29G. The pre-implantation scaffold had an area of 26.30±1.98 mm².Following the implantation, the area of the scaffold declined to20.74±1.80 mm² after 1 week, 16.41±2.44 mm² after 4 weeks and 13.82±3.88mm² after 8 weeks. The surface area of the cellulose scaffold has asignificant decrease of about 12 mm² (48%) after 8 weeks implantation(*=P<0.001; n=12-14).

For histological analysis, the following experiments were performed.

Serial 5 μm thick sections were cut, beginning at 1 mm inside thecellulose scaffold, and stained with hematoxylin-eosin (H&E) andMasson's trichrome. For immunocytochemistry, heat induced epitoperetrieval was performed at 110° C. for 12 min with citrate buffer (pH6.0). AntiCD31/PECAM1 (1:100; Novus Biologicals, NB100-2284, Oakville,ON, Canada), anti-alpha smooth muscle actin (1:1000, ab5694, abcam,Toronto, ON, Canada) and anti-CD45 (1:3000; ab10558, abcam, Toronto, ON,Canada) primary antibodies were incubated for an hour at roomtemperature. Blocking reagent (Background Sniper, Biocare, Medical,Concorde, Calif., USA) and detection system MACH 4 (Biocare Medical,Concorde, Calif., USA) were applied according to company specifications.For the evaluation of cell infiltration, extracellular matrix depositionand vascularisation (angiogenesis), micrographs were captured usingZeiss MIRAX MIDI Slide Scanner (Zeiss, Toronto, Canada) equipped with40× objective and analysed using Pannoramic Viewer (3DHISTECH Ltd.,Budapest, Hungary) and ImageJ software.

FIG. 30 shows results of biocompatibility and cell infiltration. Crosssections of representative cellulose scaffolds were stained with H&E andanti-CD45. These global views show the acute moderate-severe anticipatedforeign body reaction at 1 week (FIG. 30A), the mild chronic immune andsubsequent cleaning processes at 4 weeks (FIG. 30B) and finally, thecellulose scaffold assimilated into the native mouse tissue at 8 weeks(FIG. 30C). Higher magnification regions of interest (FIG. 30D-F), seeinset (FIG. 30A-C), allow the observation of the cell type populationwithin biomaterial assimilation processes. At 1 week, we can observepopulations of granulocytes, specifically; polymorphonuclear (PMN) andeosinophils that characterize the acute moderate to severe immuneresponse, a normal reaction to implantation procedures (FIG. 30D). At 4weeks, a decreased immune response can be observed (mild to low immuneresponse) and the population of cells within the epidermis surroundingscaffolds now contain higher levels of monocytes and lymphocytescharacterizing chronic response (FIG. 30E). Finally, at 8 weeks, theimmune response has completely resorbed with the epidermis tissue nowappearing normal (FIG. 30F). The immune response observed with H&Estaining is confirmed using anti-CD45 antibody, a well-known marker ofleukocytes (FIG. 30G-I). The population of cells within the scaffold arenow mainly macrophages, multinucleated cells and active fibroblasts.

The presence of active fibroblasts raised a question of whether thecellulose scaffold was acting as a substrate for the deposition of newextracellular matrix. This was determined using Masson's Trichromestaining of fixed cellulose scaffolds slides at each time pointfollowing implantation (FIG. 31). At 1-week post-implantation, thehistological study shows the absence of collagen structures inside thecollagen scaffold (FIG. 31A,D,G). After 4-weeks, small amounts ofcollagen begin to be deposited inside the scaffold (FIG. 31B,E,H) and by8-weeks, large amounts of collagen are clearly visible within manyscaffold cavities (FIG. 31C,F,I). The presence of active fibroblastsidentified through morphology (H&E staining, spindle shaped) andanti-alpha smooth muscle actin staining (data not shown) are completelyconsistent with the large degree of collagen deposits observed at8-weeks. The complexity of the deposited collagen network is highlightedin FIG. 311, where individual collagen fibers within the collagen matrixare visible. This is in contrast to the characteristic high density,thick, cable-like organization of collagen found in scar tissue.

Capillaries ranging from 8 to 25 μm were also identified within thescaffolds as early as 1 week post-implantation. At 4 week and 8-weekpost implantation, blood vessels and capillaries can be observedextensively within the scaffold and the surrounding dermal tissue. Weobserved blood vessels presence on the cellulose scaffold and insurrounding dermis in the macroscopic photos taken during the resection(FIG. 32A). Multiple cross sections of blood vessels, with the presenceof red blood cells (RBCs), are identified within 4 weeks of scaffoldimplantations (FIG. 32B; H&E stain). The same results are obtained 8weeks after implantation where capillaries with RBC and endothelialcells are clearly seen (FIG. 32C; Masson's Trichrome).

Example 13—Bio-Inspired and Bio-Functional Grafts for Repair of SpinalCord Injury

Processes as described herein may be used to produce sterile cellulosegrafts which retain their shape and mechanical strength. Utilizing ourin-house bulk mechanical testing apparatus, the elastic modulus of ournative cellulose grafts has been recorded at ˜2 MPa when the graft iscompressed in the direction parallel to the straight microchannels. Whenthe grafts are compressed in a direction perpendicular to themicrochannels the modulus is observed to be smaller by about an order ofmagnitude. These values are highly consistent with the elastic modulusof the dura mater and pia mater meaning that these grafts fall withinrange of the mechanical properties of much of the surrounding spinalcord tissue. FIG. 33 shows images of decellularised asparagus xylemstructures and microchannels.

Brain dissections and resections of adult rats allowed for thederivation of primary rat neurospheres. The dorsal region was cleanedexposing the medulla. Using the malleus nippers the posterior skull bonewas removed, all the way to the frontal lobe, exposing the brain asparts of the skull are removed. The brain was gently removed from theskull with the final cut of the olfactory bulbs. The brain removed wassubmerged in a petri dish filled with of dissection media on ice (MEMAlpha medium (Life Technologies Inc) 1% L-Glutamine (Life TechnologiesInc) and 1% Penicillin (Life Technologies Inc). The brain was thensectioned in the brain matrix and sections containing the hippocampus.The grey matter just lateral to the 3rd ventricle was collected in atest tube with dissection media. The grey matter tissue in thedissection media was continuously centrifuged and the supernatant wascollected. Once all the supernatant is removed the final tube wascentrifuged and the pellet was re-suspended in 2 mL of culture media(Advanced DMEM/F12 medium (Life Technologies Inc), 1% L-Glutamine (LifeTechnologies Inc) and 1% N2 supplement (CEDARLANE LABORATORIES LTD)).The re-suspended cell solution was aliquoted into 6 well ultra-lowattachment plates with 0.001% human epidermal growth factor and basicfibroblast growth factor (PEPROTECH) to allow the primary ratneurospheres to proliferate. The neurospheres were locally seeded on topof individual grafts in custom fabricated cell culture chambers. Theneurospheres were cultured and maintained for 2-weeks in a 5% CO₂incubators. The culture media was changed daily. The scaffold sampleswere fixed with 4% paraformaldehyde. The cellulose cell was stained withthe previously used protocol. The neurospheres were stained with wheatgerm agglutinin (WGA) 488 (Invitrogen) examined and with confocalfluorescence microscopy (FIG. 34A).

Following a similar protocol to that discussed in the study of Example3, decellularized vascular plant was subcutaneously implanted in mice.Histological results demonstrate that after 4 weeks implantation, thevascular structures remained intact and are apparent throughout thescaffold (FIG. 34B). Consistent with the structures host cells can beobserved through the entire 5 mm span of the cellulose scaffold.Following the successful primary results of the in vitro and in vivo,the decellularized plant scaffold was fashioned into a spinal cordinjury graft. Female Sprague Dawley rats were anesthetized withisoflurane. The overlying skin was shaved and prepped with Betadine.Under aseptic conditions and using sterilized instruments, vertebrae T7to T10 were exposed. Following the dissection of the back andintercostal muscles, a laminectomy is made at the T8 and T9. The duralis exposed with micro scissors. The T8 spinal cord is transected withmicroscissors in one clear cut motion. Any bleeding resulting from thetransection is controlled with surgifoam. The spinal cord is allowed toretract and the cellulose scaffold is moved and placed to connect thecaudal and cranial stumps (FIG. 34C). Following the scaffold placement,the Tisseel fibrin glue (Baxter) was used to secure the cellulose graft.The muscle layer of the incision is closed with 3-0 Vicryl suturematerial and the epidermis and dermis are closed with Michel clips.Buprenorphine was administered prior to closure to ensure it is activelyworking by the time the rats recover from the anesthetics.

The BBB scores were observed to increase over the course of 8 weeks.

Eight weeks post-implantation, rats (n=7) exhibited improved locomotoractivity (BBB=9.2±2.5), displaying coordinated stepping and the abilityto bear weight (FIG. 35). In addition, at 8-weeks, a second spinal cordtransection (below the graft) was performed causing BBB scores to returnto 0. Control rats (n=7, fibrin only) displayed BBB scores in the 0 to 1range. The results suggest that locomotor recovery is likely not due toreflex. Spinal cords were then dissected at 8-week and sectioned at thegraft site. Slides were stained with a combination of hematoxylin, eosinand luxol fast blue (H&E-LFB) in order to indicate myelinated neurons.Data reveals positive staining for host cells passing through themicrochannels of the graft, consistent with the improvement of locomotorfunction (FIG. 35D). Additionally, we were able to demonstrate andoptimize an MM protocol that allows observe the continuity of the spinalcord and if the graft has collapsed without sacrificing the animals. Thecranial and caudal stump interface (FIG. 35A-i, 3A-iii) can be clearlydifferentiated from the scaffold graft (FIG. 35A-ii). FIGS. 36 and 37show a global view of the spinal cord graft implanted in the T8-T9region of the spine, and ventral sections of the surrounding transectionsite, respectively. As shown in FIG. 37, green filaments can be observedsurrounding the spinal graft stretching in the ventral direction (redarrows). These filaments represent mature neurons within the transectedsite of the rat after 12 weeks in vivo. Conversely, within the controlB) organized neuro filaments cannot be observed indicating a lack ofmature filaments within the control transection site. Additionally, theHoechst stain reveals a significantly increased number of nuclei, and assuch cells, surrounding the spinal graft within the transection sitecompared to the control.

In these studies, insertion of the scaffold biomaterial between thetransected spinal cord stumps, followed by fibrin glue application andwound repair, has shown that after only 8 weeks of study, control rats(n=4, no graft) exhibited no improvement in motor function and remainedcompletely paralyzed (BBB between 0-1). Remarkably, rats (n=7)possessing an asparagus-derived implant exhibited a BBB of 9.2±2.5,demonstrating a dramatic improvement in locomotor function in thesestudies. These animals exhibit coordinated stepping and the ability tobear weight. As such, asparagus-derived implants display promise fortreating SCI in a rat model. In certain embodiments, scaffoldbiomaterials as described herein may be used for recruitingneuroprogenitor cells in damaged spinal cord tissue for improvement ofmotor function.

Example 14—Plant Decellularised Scaffold for Cutaneous Skin Graft

Mice were anesthetized using 2% Isoflurane USP-PPC (Pharmaceuticalpartners of Canada, Richmond, ON, Canada) with the eyes protected withthe application of ophthalmic liquid gel (Alco Canada In., ON, Canada).The mouse back hairs were shaved. The shaved skin was then treated witha Nair gel for a duration of two minutes. The Nair was carefully removedfrom the skin and the underlying skin was cleaned and sterilized usingENDURE 400 Scrub-Stat4 Surgical Scrub (chlorhexidine gluconate, 4%solution; Ecolab Inc., Minnesota, USA) and Soluprep (2% w/vchlorhexidine and 70% v/v isopropyl alcohol; 3M Canada, London, ON,Canada). Animal hydration was maintained, via subcutaneous injection(s.c) of 1 ml of 0.9% sodium chloride solution (Hospira, Montreal, QC,Canada). Throughout the surgical procedures strict sterility measureswere upheld for survival surgeries. A 5 mm circular skin biopsy isremoved. A rubber insulating pad with gel superglue is carefullypositioned over the biopsy while still exposing the skin biopsy. Therubber pad is then sutured to the mouse at 8 points using Surgipro IImonofilament polypropylene 6-0 (Covidien, Mass., USA). The skin graft isthen placed to replace the removed skin and sealed with a two absorbenttransparent adhesion tape. Transdermal bupivicaine 2% (as monohydrate;Chiron Compounding Pharmacy Inc., Guelph, ON, Canada) was topicallyapplied to the surgery sites to prevent infection. Additionally,buprenorphine (as HCL) (0.03 mg/ml; Chiron Compounding Pharmacy Inc.Guelph, ON, Canada) was administrated s.c. as a pain reliever. Allanimals were then carefully monitored for the following 3 days by animalcare services and received additional treatment of the samepharmacological treatments. The transparent adhesion was changed everyday and the skin graft was photographed.

FIG. 38 shows a decellularised apple hypanthium tissue processed forskin grafts. Photographs were taken after 4 days to measure the degreeof host cell infiltration during the wound healing process (FIG. 38C);At 2 weeks after scaffold implantation, the mice were euthanized usingCO₂ inhalation. The dorsal skin was carefully resected and immediatelyimmersed in PBS solution. The skin sections containing cellulosescaffolds were then photographed, cut and fixed in 10% formalin for atleast 48 hours. The samples were then kept in 70% ethanol before beingembedded in paraffin by the PALM Histology Core Facility of theUniversity of Ottawa.

Example 15—Plant Decellularised Scaffold for Bone Grafts

This study was performed to show the efficiency of biomaterials asdescribed herein for bone regeneration. Here, a rat critical sizecalvarial defect was used to demonstrate that a cellulose scaffold maysuccessfully support bone regeneration in a 5 mm circular defect.

Sprague Dawley rats were anesthetized with isoflurane in oxygen andreceived subcutaneous injections of buprenorphine and sterile salineprior to surgical procedure. The rats were shaved from the bridge of thesnout between the eyes to the cauda end of the calvarium, the eyes wereprotected by applying ophthalmic liquid gel. Rats were placed in astereotaxic frame, secured by ear bars, over a water-heated warm pad. Anincision (1.5 cm) was made down to the periosteum over the scalp fromthe nasal bone to just caudal to the middle sagittal crest (bregma). Theperiosteum was divided down the sagittal midline and dissected. Thecalvarium was drilled in the right (or left) lateral parietal bone witha 5 mm trephine and a surgical drill. The score bone was dethatched fromthe dura, leaving 5 mm circular defects on rat's cranium. The defectswere cautiously washed with sterile normal saline and a 5 mm diametercylindrical (1 mm thick) cellulose scaffold (FIG. 39A) was implanted inthe defects (FIG. 39B). The skin was closed by suturing skin layers. Therats were euthanized at 4 weeks post-surgery using carbon dioxideinhalation and exsanguination, and the cellulose scaffold were recoveredalong with the surrounding bone tissue (FIG. 39C) for histologicalanalysis (FIG. 39D). Tissues were fixed in a buffered formalin solutionand dehydrated in ethanol prior to be embedded in methyl methacrylate.Various 5 μm thick samples were stained with hematoxylin/eosin tohighlight the presence cellular components (nuclei and cytoplasm) (FIG.40D). To quantitatively measure the efficiency of the cellulosescaffolds, we have used a scoring method shown in table 1—quantitativehistological scoring parameter (Kretlow et al. 2010) was used.

TABLE 1 Quantitative Histological Scoring Parameter (Kretlow et al.,2010) Description Score Hard tissue response at scaffold-bone interfaceDirect bone-to-implant contact without soft interlayer 4 Remodelinglacuna with osteoblasts and/or osteoclasts at surface 3 Majority ofimplant is surrounded by fibrous tissue capsule 2 Unorganized fibroustissue (majority of tissue is not arranged as 1 capsule) Inflammationmarked by an abundance of inflammatory cells and 0 poorly organizedtissue Hard tissue response within the pores of the scaffold Tissue inpores in mostly bone 4 Tissue in pores consists of some bone withinmature, dense fibrous 3 tissue and/or a few inflammatory responseelements Tissues in pores is mostly immature fibrous tissue (with orwithout 2 bone) with blood vessels and young fibroblasts invading thespace with few macrophages present Tissues in pores consists mostly ofinflammatory cells and 1 connective tissue components in between (withor without bone) or the majority of the pores are empty or filled withfluid Tissue in pores is dense and exclusively of inflammatory type (no0 bone present)

In the experiments shown in FIG. 39, plant derived cellulose scaffoldswere assessed for bone grafts. As described, each cylindrical (5 mmdiameter, 1 mm thick) implant was measured prior to the implantation forscaffold area comparison (FIG. 39A). Cellulose scaffold implants wereimplanted into the rat skull defects and positioned to remain within theskull defect. The skin was then positioned over the graft and sutured soas to keep the scaffold in place (FIG. 39B). The scaffold andsurrounding bone tissues were isolated 4 weeks after the implantationand macroscopic pictures were taken (FIG. 39C). The isolated tissue wasthen decalcified and processed/embedded in paraffin. Serial 5 μm thicksections were cut, beginning at 1 mm inside the cellulose scaffold, andstained with hematoxylin-eosin (H&E) (FIG. 39D). For the evaluation ofbone regeneration, micrographs were captured using Zeiss MIRAX MIDISlide Scanner (Zeiss, Toronto, Canada) equipped with 40× objective andanalysed using Panoramic Viewer (3DHISTECH Ltd., Budapest, Hungary) andImageJ software.

Histological results show a direct bone to scaffold contact at theinterface of the defect and the biomaterial scaffolds.

Example 16—Example Forms of Scaffold Biomaterials

FIGS. 40A to 40F show different example formulations, physicalproperties and functionalizations of cellulose-based scaffoldbiomaterials. FIG. 40A shows that cellulose may be used as block cutinto different shapes. FIG. 40B shows that cellulose may be dehydratedand ground into a powdered form. FIG. 40B also shows that if thecellulose contains carboxymethylcellulose, it may easily be crosslinkedwith citric acid and heat. FIG. 40C shows that the powdered form of thecellulose may be rehydrated to a desired consistency to produce a gel(FIG. 40D) or a paste (FIGS. 40E, 40F).

Example 17—Survival Rate Following Implantation

FIG. 41A is a graph showing the experimental survival rate of mice(n=190) and rats (n=12) following the implantation of the biomaterial(from various sources) at 1 week, 4 weeks and 8 weeks post-implantation.FIG. 41B shows the rate of biomaterial rejection at these same timepoints as in FIG. 41A. All animals (mice and rats) survived thebiomaterial implantation, and all survive the complete duration of eachtrial and none showed signs of implant rejection in these experiments.

Example 18—Examples of Plant and Fungi Tissues

Different taxonomy plant systems are used in plant classification andseveral versions of these systems exist (ex: Cronquist system and APGsystems).

In experiments as described herein, by using a wide range of plantswhich are classified in different plant groups, families, genera andspecies, our data indicates that a wide variety of plants may be used asin the preparation of scaffold biomaterials.

Generally speaking, the plant kingdom is divided in four major groups:

-   -   Flowering plants (Angiosperms);    -   Conifers, cycads and allies (Gymnosperms);    -   Ferns and fern allies (Pteridophytes);    -   Mosses and liverworts (Bryophytes).

These four major groups contain many plant families which are divided inmany genera that are also divided in species. The following is a list ofthe major plant families from which cellulose scaffolds may begenerated:

Acanthaceae, Achariaceae, Achatocarpaceae, Acoraceae, Acrobolbaceae,Actinidiaceae, Adelanthaceae, Adoxaceae, Aextoxicaceae, Aizoaceae,Akaniaceae, Alismataceae, Allisoniaceae, Alseuosmiaceae,Alstroemeriaceae, Altingiaceae, Amaranthaceae, Amaryllidaceae,Amblystegiaceae, Amborellaceae, Anacampserotaceae, Anacardiaceae,Anarthriaceae, Anastrophyllaceae, Ancistrocladaceae, Andreaeaceae,Andreaeobryaceae, Anemiaceae, Aneuraceae, Anisophylleaceae, Annonaceae,Antheliaceae, Anthocerotaceae, Aphanopetalaceae, Aphloiaceae, Apiaceae,Apleniaceae, Apocynaceae, Apodanthaceae, Aponogetonaceae, Aquifoliaceae,Araceae, Araliaceae, Araucariaceae, Archidiaceae, Arecaceae,Argophyllaceae, Aristolochiaceae, Arnelliaceae, Asparagaceae,Aspleniaceae, Asteliaceae, Asteropeiaceae, Atherospermataceae,Athyriaceae, Aulacomniaceae, Austrobaileyaceae, Aytoniaceae,Balanopaceae, Balanophoraceae, Balantiopsaceae, Balsaminaceae,Barbeuiaceae, Barbeyaceae, Bartramiaceae, Basellaceae, Bataceae,Begoniaceae, Berberidaceae, Berberidopsidaceae, Betulaceae,Biebersteiniaceae, Bignoniaceae, Bixaceae, Blandfordiaceae, Blasiaceae,Blechnaceae, Bonnetiaceae, Boraginaceae, Boryaceae, Brachytheciaceae,Brassicaceae, Brevianthaceae, Bromeliaceae, Bruchiaceae, Brunelliaceae,Bruniaceae, Bryaceae, Bryobartramiaceae, Bryoxiphiaceae, Burmanniaceae,Burseraceae, Butomaceae, Buxaceae, Buxbaumiaceae, Byblidaceae,Cabombaceae, Cactaceae, Calceolariaceae, Calomniaceae, Calophyllaceae,Calycanthaceae, Calyceraceae, Calymperaceae, Calypogeiaceae,Campanulaceae, Campynemataceae, Canellaceae, Cannabaceae, Cannaceae,Capparaceae, Caprifoliaceae, Cardiopteridaceae, Caricaceae,Carlemanniaceae, Caryocaraceae, Caryophyllaceae, Casuarinaceae,Catagoniaceae, Catoscopiaceae, Celastraceae, Centrolepidaceae,Centroplacaceae, Cephalotaceae, Cephaloziaceae, Cephaloziellaceae,Ceratophyllaceae, Cercidiphyllaceae, Chaetophyllopsaceae,Chloranthaceae, Chonecoleaceae, Chrysobalanaceae, Cibotiaceae,Cinclidotaceae, Circaeasteraceae, Cistaceae, Cleomaceae, Clethraceae,Cleveaceae, Climaciaceae, Clusiaceae, Colchicaceae, Columelliaceae,Combretaceae, Commelinaceae, Compositae, Connaraceae, Conocephalaceae,Convolvulaceae, Coriariaceae, Cornaceae, Corsiaceae, Corsiniaceae,Corynocarpaceae, Costaceae, Crassulaceae, Crossosomataceae,Cryphaeaceae, Ctenolophonaceae, Cucurbitaceae, Culcitaceae, Cunoniaceae,Cupressaceae, Curtisiaceae, Cyatheaceae, Cycadaceae, Cyclanthaceae,Cymodoceaceae, Cynomoriaceae, Cyperaceae, Cyrillaceae, Cyrtopodaceae,Cystodiaceae, Cystopteridaceae, Cytinaceae, Daltoniaceae,Daphniphyllaceae, Dasypogonaceae, Datiscaceae, Davalliaceae,Degeneriaceae, Dendrocerotaceae, Dennstaedtiaceae, Diapensiaceae,Dichapetalaceae, Dicksoniaceae, Dicnemonaceae, Dicranaceae,Didiereaceae, Dilleniaceae, Dioncophyllaceae, Dioscoreaceae,Dipentodontaceae, Diphysciaceae, Diplaziopsidaceae, Dipteridaceae,Dipterocarpaceae, Dirachmaceae, Disceliaceae, Ditrichaceae,Doryanthaceae, Droseraceae, Drosophyllaceae, Dryopteridacae,Dryopteridaceae, Ebenaceae, Ecdeiocoleaceae, Echinodiaceae,Elaeagnaceae, Elaeocarpaceae, Elatinaceae, Emblingiaceae, Encalyptaceae,Entodontaceae, Ephedraceae, Ephemeraceae, Equisetaceae, Ericaceae,Eriocaulaceae, Erpodiaceae, Erythroxylaceae, Escalloniaceae,Eucommiaceae, Euphorbiaceae, Euphroniaceae, Eupomatiaceae, Eupteleaceae,Eustichiaceae, Exormothecaceae, Fabroniaceae, Fagaceae, Fissidentaceae,Flacourtiaceae, Flagellariaceae, Fontinalaceae, Fossombroniaceae,Fouquieriaceae, Frankeniaceae, Funariaceae, Garryaceae,Geissolomataceae, Gelsemiaceae, Gentianaceae, Geocalycaceae,Geraniaceae, Gerrardinaceae, Gesneriaceae, Gigaspermaceae, Ginkgoaceae,Gisekiaceae, Gleicheniaceae, Gnetaceae, Goebeliellaceae, Gomortegaceae,Goodeniaceae, Goupiaceae, Grimmiaceae, Grossulariaceae, Grubbiaceae,Guamatelaceae, Gunneraceae, Gymnomitriaceae, Gyrostemonaceae,Gyrothyraceae, Haemodoraceae, Halophytaceae, Haloragaceae,Hamamelidaceae, Hanguanaceae, Haplomitriaceae, Haptanthaceae,Hedwigiaceae, Heliconiaceae, Helicophyllaceae, Helwingiaceae,Herbertaceae, Hernandiaceae, Himantandraceae, Hookeriaceae, Huaceae,Humiriaceae, Hydatellaceae, Hydnoraceae, Hydrangeaceae,Hydrocharitaceae, Hydroleaceae, Hydrostachyaceae, Hylocomiaceae,Hymenophyllaceae, Hymenophytaceae, Hypericaceae, Hypnaceae,Hypnodendraceae, Hypodematiaceae, Hypopterygiaceae, Hypoxidaceae,Icacinaceae, Iridaceae, Irvingiaceae, Isoëtaceae, teaceae,Ixioliriaceae, Ixonanthaceae, Jackiellaceae, Joinvilleaceae, Jubulaceae,Jubulopsaceae, Juglandaceae, Juncaceae, Juncaginaceae, Jungermanniaceae,Kirkiaceae, Koeberliniaceae, Krameriaceae, Lacistemataceae,Lactoridaceae, Lamiaceae, Lanariaceae, Lardizabalaceae, Lauraceae,Lecythidaceae, Leguminosae, Lejeuneaceae, Lembophyllaceae,Lentibulariaceae, Lepicoleaceae, Lepidobotryaceae, Lepidolaenaceae,Lepidoziaceae, Leptodontaceae, Lepyrodontaceae, Leskeaceae,Leucodontaceae, Leucomiaceae, Liliaceae, Limeaceae, Limnanthaceae,Linaceae, Linderniaceae, Lindsaeaceae, Loasaceae, Loganiaceae,Lomariopsidaceae, Lonchitidaceae, Lophiocarpaceae, Lophocoleaceae,Lophopyxidaceae, Lophoziaceae, Loranthaceae, Lowiaceae, Loxsomataceae,Lunulariaceae, Lycopodiaceae, Lygodiaceae, Lythraceae, Magnoliaceae,Makinoaceae, Malpighiaceae, Malvaceae, Marantaceae, Marattiaceae,Marcgraviaceae, Marchantiaceae, Marsileaceae, Martyniaceae,Mastigophoraceae, Matoniaceae, Mayacaceae, Meesiaceae, Melanthiaceae,Melastomataceae, Meliaceae, Melianthaceae, Menispermaceae,Menyanthaceae, Mesoptychiaceae, Metaxyaceae, Meteoriaceae,Metteniusaceae, Metzgeriaceae, Microtheciellaceae, Misodendraceae,Mitrastemonaceae, Mitteniaceae, Mizutaniaceae, Mniaceae, Molluginaceae,Monimiaceae, Monocarpaceae, Monocleaceae, Monosoleniaceae, Montiaceae,Montiniaceae, Moraceae, Moringaceae, Muntingiaceae, Musaceae,Myodocarpaceae, Myricaceae, Myriniaceae, Myristicaceae, Myrothamnaceae,Myrtaceae, Myuriaceae, Nartheciaceae, Neckeraceae, Nelumbonaceae,Neotrichocoleaceae, Nepenthaceae, Nephrolepidaceae, Neuradaceae,Nitrariaceae, Nothofagaceae, Notothyladaceae, Nyctaginaceae,Nymphaeaceae, Ochnaceae, Octoblepharaceae, Oedipodiaceae, Olacaceae,Oleaceae, Oleandraceae, Onagraceae, Oncothecaceae, Onocleaceae,Ophioglossaceae, Opiliaceae, Orchidaceae, Orobanchaceae,Orthorrhynchiaceae, Orthotrichaceae, Osmundaceae, Oxalidaceae,Oxymitraceae, Paeoniaceae, Pallaviciniaceae, Pandaceae, Pandanaceae,Papaveraceae, Paracryphiaceae, Passifloraceae, Paulowniaceae,Pedaliaceae, Pelliaceae, Penaeaceae, Pennantiaceae, Pentadiplandraceae,Pentaphragmataceae, Pentaphylacaceae, Penthoraceae, Peraceae,Peridiscaceae, Petenaeaceae, Petermanniaceae, Petrosaviaceae,Phellinaceae, Philesiaceae, Philydraceae, Phrymaceae, Phyllanthaceae,Phyllodrepaniaceae, Phyllogoniaceae, Phyllonomaceae, Physenaceae,Phytolaccaceae, Picramniaceae, Picrodendraceae, Pilotrichaceae,Pinaceae, Piperaceae, Pittosporaceae, Plagiochilaceae, Plagiogyriaceae,Plagiotheciaceae, Plantaginaceae, Platanaceae, Pleurophascaceae,Pleuroziaceae, Pleuroziopsaceae, Plocospermataceae, Plumbaginaceae,Poaceae, Podocarpaceae, Podostemaceae, Polemoniaceae, Polygalaceae,Polygonaceae, Polypodiaceae, Polytrichaceae, Pontederiaceae,Porellaceae, Portulacaceae, Posidoniaceae, Potamogetonaceae, Pottiaceae,Primulaceae, Prionodontaceae, Proteaceae, Pseudoditrichaceae,Pseudolepicoleaceae, Psilotaceae, Pteridaceae, Pterigynandraceae,Pterobryaceae, Ptilidiaceae, Ptychomitriaceae, Ptychomniaceae,Putranjivaceae, Quillajaceae, Racopilaceae, Radulaceae, Rafflesiaceae,Ranunculaceae, Rapateaceae, Regmatodontaceae, Resedaceae, Restionaceae,Rhabdodendraceae, Rhabdoweisiaceae, Rhachidosoraceae, Rhachitheciaceae,Rhacocarpaceae, Rhamnaceae, Rhipogonaceae, Rhizogoniaceae,Rhizophoraceae, Ricciaceae, Riellaceae, Rigodiaceae, Roridulaceae,Rosaceae, Rousseaceae, Rubiaceae, Ruppiaceae, Rutaceae, Rutenbergiaceae,Sabiaceae, Saccolomataceae, Salicaceae, Salvadoraceae, Salviniaceae,Santalaceae, Sapindaceae, Sapotaceae, Sarcobataceae, Sarcolaenaceae,Sarraceniaceae, Saururaceae, Saxifragaceae, Scapaniaceae,Scheuchzeriaceae, Schisandraceae, Schistochilaceae, Schistostegaceae,Schizaeaceae, Schlegeliaceae, Schoepfiaceae, Sciadopityaceae,Scorpidiaceae, Scrophulariaceae, Selaginellaceae, Seligeriaceae,Sematophyllaceae, Serpotortellaceae, Setchellanthaceae, Simaroubaceae,Simmondsiaceae, Siparunaceae, Sladeniaceae, Smilacaceae, Solanaceae,Sorapillaceae, Sphaerocarpaceae, Sphaerosepalaceae, Sphagnaceae,Sphenocleaceae, Spiridentaceae, Splachnaceae, Splachnobryaceae,Stachyuraceae, Staphyleaceae, Stegnospermataceae, Stemonaceae,Stemonuraceae, Stereophyllaceae, Stilbaceae, Strasburgeriaceae,Strelitziaceae, Stylidiaceae, Styracaceae, Surianaceae, Symplocaceae,Takakiaceae, Talinaceae, Tamaricaceae, Tapisciaceae, Targioniaceae,Taxaceae, Tecophilaeaceae, Tectariaceae, Tetrachondraceae,Tetramelaceae, Tetrameristaceae, Tetraphidaceae, Thamnobryaceae,Theaceae, Theliaceae, Thelypteridaceae, Thomandersiaceae, Thuidiaceae,Thurniaceae, Thymelaeaceae, Thyrsopteridaceae, Ticodendraceae,Timmiaceae, Tofieldiaceae, Torricelliaceae, Tovariaceae, Trachypodaceae,Treubiaceae, Trichocoleaceae, Trichotemnomataceae, Trigoniaceae,Trimeniaceae, Triuridaceae, Trochodendraceae, Tropaeolaceae, Typhaceae,Ulmaceae, Urticaceae, Vahliaceae, Vandiemeniaceae, Velloziaceae,Verbenaceae, Vetaformaceae, Violaceae, Viridivelleraceae, Vitaceae,Vivianiaceae, Vochysiaceae, Wardiaceae, Welwitschiaceae,Wiesnerellaceae, Winteraceae, Woodsiaceae, Xanthorrhoeaceae,Xeronemataceae, Xyridaceae, Zamiaceae, Zingiberaceae, Zosteraceae,Zygophyllaceae.

Because of a new classification, some groups of algae are no longerclassified within the plant kingdom. These algae are, nevertheless,candidates for cellulose scaffold production as described herein. Thefungi Kingdom has members which contain, for example, a cell wall madeof cellulose. Algae are now classified in the protista Kingdom; however,it will be understood that in this disclosure, algae are intended to beencompassed by the term “plants” as used herein. Suitable algae mayinclude:

-   -   Algae: plant-like single or multi-celled organisms;    -   Green algae: Spirogyra, Ulva, Chlamydomonas, Volvox;    -   Red algae: Porphyra, Rotalgen;    -   Brown algae: Laminaria, Nereocystis;    -   Water molds: Saprolegnia; and/or    -   Phylum Ciliata: Paramecium, Vorticella.

It has also been experimentally demonstrated that chitin is a suitablescaffold which may be used in scaffold biomaterials as described hereinusing protocols as described herein. The fungi Kingdom is classified asfollows:

-   -   Sac-fungi: Agaricus (mushroom), Ustilago (smut), and Puccinia        (rust fungus);    -   Zygote-forming fungi: Mucor, Rhizopus (the bread mould) and        Albugo;    -   Club fungi: Agaricus (mushroom), Ustilago (smut), and Puccinia        (rust fungus); and    -   Imperfect fungi: Alternaria, Colletotrichum and Trichoderma.

Such fungi also represent suitable candidates for obtainingdecellularised fungal tissues as described hereinabove.

One or more illustrative embodiments have been described by way ofexample. It will be understood to persons skilled in the art that anumber of variations and modifications can be made without departingfrom the scope of the invention as defined in the claims.

REFERENCES

-   1. Saini M. Implant biomaterials: A comprehensive review. World J    Clin Cases. 2015; 3: 52. doi:10.12998/wjcc.v3.i1.52-   2. Pashuck E T, Stevens M M. STATE OF THE ART REVIEW Designing    Regenerative Biomaterial Therapies for the Clinic. Sci Transl Med.    2012; 4.-   3. Athanasiou K A, Reddi A H, Guldberg R E, Revell C M. Special    section. 2012; 338: 921-927.-   4. Kar M, Vernon Shih Y-R, Velez D O, Cabrales P, Varghese S.    Poly(ethylene glycol) hydrogels with cell cleavable groups for    autonomous cell delivery. Biomaterials. 2016; 77: 186-97.    doi:10.1016/j.biomaterials.2015.11.018-   5. Gu L, Mooney D J. Biomaterials and emerging anticancer    therapeutics: engineering the microenvironment. Nat Rev Cancer.    Nature Publishing Group, a division of Macmillan Publishers Limited.    All Rights Reserved; 2015; 16: 56-66. doi:10.1038/nrc.2015.3-   6. Maurer M, Rohrnbauer B, Feola A, Deprest J, Mazza E. Prosthetic    Meshes for Repair of Hernia and Pelvic Organ Prolapse: Comparison of    Biomechanical Properties. Materials (Basel). Multidisciplinary    Digital Publishing Institute; 2015; 8: 2794-2808.    doi:10.3390/ma8052794-   7. Mao A S, Mooney D J. Regenerative medicine: Current therapies and    future directions. Proc Natl Acad Sci. 2015; 112: 201508520.    doi:10.1073/pnas.1508520112-   8. Hsu S-H, Hsieh P-S. Self-assembled adult adipose-derived stem    cell spheroids combined with biomaterials promote wound healing in a    rat skin repair model. Wound Repair Regen. 23: 57-64.    doi:10.1111/wrr.12239-   9. Guillaume O, Park J, Monforte X, Gruber-Blum S, Redl H,    Petter-Puchner A, et al. Fabrication of silk mesh with enhanced    cytocompatibility: preliminary in vitro investigation toward    cell-based therapy for hernia repair. J Mater Sci Mater Med. 2016;    27: 37. doi:10.1007/s10856-015-5648-3-   10. Soto-Gutierrez A, Zhang L, Medberry C, Fukumitsu K, Faulk D,    Jiang H, et al. A whole-organ regenerative medicine approach for    liver replacement. Tissue Eng Part C Methods. Mary Ann Liebert, Inc.    140 Huguenot Street, 3rd Floor New Rochelle, N.Y. 10801 USA; 2011;    17: 677-86. doi:10.1089/ten.TEC.2010.0698-   11. Badylak S F, Taylor D, Uygun K. Whole-Organ Tissue Engineering:    Decellularization and Recellularization of Three-Dimensional Matrix    Scaffolds. Annual Reviews; 2011; Available:    http://www.annualreviews.org/doi/abs/10.1146/annurev-bioeng-071910-124743-   12. Baptista P M, Orlando G, Mirmalek-Sani S-H, Siddiqui M, Atala A,    Soker S. Whole organ decellularization—a tool for bioscaffold    fabrication and organ bioengineering. Conf Proc. Annu Int Conf IEEE    Eng Med Biol Soc IEEE Eng Med Biol Soc Annu Conf. 2009; 2009:    6526-9. doi:10.1109/IEMBS.2009.5333145-   13. Baptista P M, Siddiqui M M, Lozier G, Rodriguez S R, Atala A,    Soker S. The use of whole organ decellularization for the generation    of a vascularized liver organoid. Hepatology. 2011; 53: 604-617.    doi:10.1002/hep.24067-   14. Ott H C, Matthiesen T S, Goh S K, Black L D, Kren S M, Netoff T    I, et al. Perfusion-decellularized matrix: using nature's platform    to engineer a bioartificial heart. Nat Med. 2008; 14: 213-21.    doi:10.1038/nm1684-   15. Song J J, Ott H C. Organ engineering based on decellularized    matrix scaffolds. Trends Mol Med. Elsevier Ltd; 2011; 17: 424-32.    doi:10.1016/j.molmed.2011.03.005-   16. Badylak S F. The extracellular matrix as a biologic scaffold    material. Biomaterials. 2007; 28: 3587-3593.    doi:10.1016/j.biomaterials.2007.04.043-   17. Lv S, Dudek D M, Cao Y, Balamurali M M, Gosline J, Li H.    Designed biomaterials to mimic the mechanical properties of muscles.    Nature. 2010; 465: 69-73. doi:10.1038/nature09024-   18. Campoli G, Borleffs M S, Amin Yavari S, Wauthle R, Weinans H,    Zadpoor a. a. Mechanical properties of open-cell metallic    biomaterials manufactured using additive manufacturing. Mater Des.    2013; 49: 957-965. doi:10.1016/j.matdes.2013.01.071-   19. Anseth K S, Bowman C N, Brannon-Peppas L. Mechanical properties    of hydrogels and their experimental determination. Biomaterials.    1996; 17: 1647-1657. doi:10.1016/0142-9612(96)87644-7-   20. Zhao R, Sider K L, Simmons C a. Measurement of layer-specific    mechanical properties in multilayered biomaterials by micropipette    aspiration. Acta Biomater. 2011; 7: 1220-1227.    doi:10.1016/j.actbio.2010.11.004-   21. Chen Q, Liang S, Thouas G a. Elastomeric biomaterials for tissue    engineering. Prog Polym Sci. 2013; 38: 584-671.    doi:10.1016/j.progpolymsci.2012.05.003-   22. Guzman R C de, Merrill M R, Richter J R, Hamzi R I,    Greengauz-Roberts O K, Van Dyke M E. Mechanical and biological    properties of keratose biomaterials. Biomaterials. 2011; 32:    8205-17. doi:10.1016/j.biomaterials.2011.07.054-   23. Staiger M P, Pietak A M, Huadmai J, Dias G. Magnesium and its    alloys as orthopedic biomaterials: A review. Biomaterials. 2006; 27:    1728-1734. doi:10.1016/j.biomaterials.2005.10.003-   24. Bagno A, Di Bello C. Surface treatments and roughness properties    of Ti-based biomaterials. J Mater Sci Mater Med. 2004; 15: 935-49.    doi:10.1023/B:JMSM.0000042679.28493.7f-   25. Tibbitt M W, Anseth K S. Dynamic Microenvironments: The Fourth    Dimension. 2012; 4: 1-5.-   26. Lemons J E, Lucas L C. Properties of biomaterials. J    Arthroplasty. 1986; 1: 143-147. doi:10.1016/S0883-5403(86)80053-5-   27. Modulevsky D J, Lefebvre C, Haase K, Al-Rekabi Z, Pelling A E.    Apple Derived Cellulose Scaffolds for 3D Mammalian Cell Culture.    Kerkis I, editor. PLoS One. 2014; 9: e97835. doi:    10.1371/journal.pone.0097835-   28. Tibbitt M W, Anseth K S. Hydrogels as extracellular matrix    mimics for 3D cell culture. Biotechnol Bioeng. 2009; 103: 655-63.    doi:10.1002/bit.22361-   29. Vacanti J P, Lal B, Grad O, Darling E M, Hu J C, Wiesmann H P,    et al. Special section. 2012; 338: 921-926.-   30. Why Organ, Eye and Tissue Donation? In: U.S. Department of    Health and Human Services [Internet]. Available:    http://www.organdonor.gov/index.html-   31. Sterling J A, Guelcher S A. Biomaterial scaffolds for treating    osteoporotic bone. Curr Osteoporos Rep. 2014; 12: 48-54.    doi:10.1007/s11914-014-0187-2-   32. Abou Neel E A, Chrzanowski W, Salih V M, Kim H-W, Knowles J C.    Tissue engineering in dentistry. J Dent. 2014; 42: 915-28.    doi:10.1016/j.jdent.2014.05.008-   33. Shue L, Yufeng Z, Mony U. Biomaterials for periodontal    regeneration: a review of ceramics and polymers. Biomatter. 2:    271-7. doi:10.4161/biom.22948-   34. O'Brien F J. Biomaterials & scaffolds for tissue engineering.    Mater Today. 2011; 14: 88-95. doi:10.1016/S1369-7021(11)70058-X-   35. Bhardwaj N, Devi D, Mandal B B. Tissue-engineered cartilage: the    crossroads of biomaterials, cells and stimulating factors. Macromol    Biosci. 2015; 15: 153-82. doi: 10.1002/mabi.201400335-   36. Metcalfe A D, Ferguson M W J. Tissue engineering of replacement    skin: the crossroads of biomaterials, wound healing, embryonic    development, stem cells and regeneration. J R Soc Interface. 2007;    4: 413-37. doi:10.1098/rsif.2006.0179-   37. Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaeri T, et    al. Vascularized and functional human liver from an iPSC-derived    organ bud transplant. Nature. Nature Publishing Group, a division of    Macmillan Publishers Limited. All Rights Reserved; 2013; 499: 481-4.    doi:10.1038/nature12271-   38. Mannoor M S, Jiang Z, James T, Kong Y L, Malatesta K A, Soboyejo    W O, et al. 3D printed bionic ears. Nano Lett. American Chemical    Society; 2013; 13: 2634-9. doi:10.1021/n14007744-   39. Raya-Rivera A M, Esquiliano D, Fierro-Pastrana R, Lopez-Bayghen    E, Valencia P, Ordorica-Flores R, et al. Tissue-engineered    autologous vaginal organs in patients: a pilot cohort study. Lancet    (London, England). Elsevier; 2014; 384: 329-36.    doi:10.1016/S0140-6736(14)60542-0-   40. Salzberg C A. Nonexpansive immediate breast reconstruction using    human acellular tissue matrix graft (AlloDerm). Ann Plast Surg.    2006; 57: 1-5. doi:10.1097/01.sap.0000214873.13102.9f-   41. Lee D K. Achilles Tendon Repair with Acellular Tissue Graft    Augmentation in Neglected Ruptures. J Foot Ankle Surg. 2007; 46:    451-455. doi:10.1053/j.jfas.2007.05.007-   42. Cornwell K G, Landsman A, James K S. Extracellular Matrix    Biomaterials for Soft Tissue Repair. Clin Podiatr Med Surg. 2009;    26: 507-523. doi:10.1016/j.cpm.2009.08.001-   43. Ren X, Moser P T, Gilpin S E, Okamoto T, Wu T, Tapias L F, et    al. Engineering pulmonary vasculature in decellularized rat and    human lungs. Nat Biotechnol. 2015; 33: 1097-102.    doi:10.1038/nbt.3354-   44. Guyette J P, Charest J, Mills R W, Jank B, Moser P T, Gilpin S    E, et al. Bioengineering Human Myocardium on Native Extracellular    Matrix. Circ Res. 2015; CIRCRESAHA.115.306874-.    doi:10.1161/CIRCRESAHA.115.306874-   45. Raya-Rivera A, Esquiliano D R, Yoo J J, Lopez-Bayghen E, Soker    S, Atala A. Tissue-engineered autologous urethras for patients who    need reconstruction: an observational study. Lancet (London,    England). 2011; 377: 1175-82. doi:10.1016/50140-6736(10)62354-9-   46. Atala A, Bauer S B, Soker S, Yoo J J, Retik A B.    Tissue-engineered autologous bladders for patients needing    cystoplasty. Lancet. 2006; 367: 1241-6.    doi:10.1016/S0140-6736(06)68438-9-   47. Hattori N. Cerebral organoids model human brain development and    microcephaly. Mov Disord. Nature Publishing Group; 2014; 29:    185-185. doi:10.1002/mds.25740-   48. Gottenbos B, Busscher H J, Van Der Mei H C, Nieuwenhuis P.    Pathogenesis and prevention of biomaterial centered infections. J    Mater Sci Mater Med. 2002; 13: 717-722. doi: 10.1023/A:    1016175502756-   49. Bohner M. Resorbable biomaterials as bone graft substitutes.    Mater Today. 2010; 13: 24-30. doi:10.1016/S1369-7021(10)70014-6-   50. Ratner B D, Hoffman A S, Schoen F J, Lemons J E. Biomaterials    science: an introduction to materials in medicine. Chemical    Engineering. 2004.-   51. Bae H, Puranik A S, Gauvin R, Edalat F, Peppas N A,    Khademhosseini A. Building Vascular Networks. 2012; 4: 1-6.-   52. Dong W, Hou L, Li T, Gong Z, Huang H, Wang G, et al. A Dual Role    of Graphene Oxide Sheet Deposition on Titanate Nanowire Scaffolds    for Osteo-implantation: Mechanical Hardener and Surface Activity    Regulator. Sci Rep. Nature Publishing Group; 2015; 5: 18266.    doi:10.1038/srep18266-   53. Zhou L, Pomerantseva I, Bassett E K, Bowley C M, Zhao X, Bichara    D a, et al. Engineering ear constructs with a composite scaffold to    maintain dimensions. Tissue Eng Part A. 2011; 17: 1573-1581.    doi:10.1089/ten.tea.2010.0627-   54. Temenoff J S, Mikos A G. Injectable biodegradable materials for    orthopedic tissue engineering. Biomaterials. 2000; 21: 2405-2412.    doi:10.1016/S0142-9612(00)00108-3-   55. Comprehensive Biomaterials: Online Version, Volume 1 [Internet].    Newnes; 2011. Available:    https://books.google.com/books?id=oa8YpRsD1kkC&pgis=1-   56. Bao G, Suresh S. Cell and molecular mechanics of biological    materials. Nat Mater. 2003; 2: 715-25. doi:10.1038/nmat1001-   57. Place E S, Evans N D, Stevens M M. Complexity in biomaterials    for tissue engineering. Nat Mater. Nature Publishing Group; 2009; 8:    457-470. doi:10.1038/nmat2441-   58. Pomerantseva I, Bichara D A, Tseng A, Cronce M J, Cervantes T M,    Kimura A M, et al. Ear-Shaped Stable Auricular Cartilage Engineered    from Extensively Expanded Chondrocytes in an Immunocompetent    Experimental Animal Model. Tissue Eng Part A. 2015; 00:    ten.tea.2015.0173. doi:10.1089/ten.tea.2015.0173-   59. Xu J-W, Johnson T S, Motarjem P M, Peretti G M, Randolph M A,    Yaremchuk M J. Tissue-engineered flexible ear-shaped cartilage.    Plast Reconstr Surg. 2005; 115: 1633-41. Available:    http://www.ncbi.nlm.nih.gov/pubmed/15861068-   60. Shieh S-J, Terada S, Vacanti J P. Tissue engineering auricular    reconstruction: in vitro and in vivo studies. Biomaterials. 2004;    25: 1545-57. Available: http://www.ncbi.nlm.nih.gov/pubmed/14697857-   61. Neumeister M W, Wu T, Chambers C. Vascularized tissue-engineered    ears. Plast Reconstr Surg. 2006; 117: 116-22. Available:    http://www.ncbi.nlm.nih.gov/pubmed/16404257-   62. Isogai N, Asamura S, Higashi T, Ikada Y, Morita S, Hillyer J, et    al. Tissue engineering of an auricular cartilage model utilizing    cultured chondrocyte-poly(L-lactide-epsilon-caprolactone) scaffolds.    Tissue Eng. 10: 673-87. doi:10.1089/1076327041348527-   63. Cervantes T M, Bassett E K, Tseng A, Kimura A, Roscioli N,    Randolph M a, et al. Design of composite scaffolds and    three-dimensional shape analysis for tissue-engineered ear. J R Soc    Interface. 2013; 10: 20130413. doi:10.1098/rsif.2013.0413-   64. Liao H T, Zheng R, Liu W, Zhang W J, Cao Y, Zhou G.    Prefabricated, Ear-Shaped Cartilage Tissue Engineering by    Scaffold-Free Porcine Chondrocyte Membrane. Plast Reconstr Surg.    2015; 135: 313-321. doi:10.1097/PRS.0000000000001105-   65. Lee J-S. 3D printing of composite tissue with complex shape    applied to ear regeneration. Biofabrication. 2014; 6. Available:    http://resolver.scholarsportal.info/resolve/17585082/v06i0002/024103_3    poctwcsater.xml-   66. Pértile RAN, Moreira S, Gil R M, Correia A, Guãrdao L. Bacterial    Cellulose: Long-Term Biocompatibility Studies. J Biomater Sci Polym    Ed. 2012; 23: 1339-1354.-   67. Entcheva E, Bien H, Yin L, Chung C Y, Farrell M, Kostov Y.    Functional cardiac cell constructs on cellulose-based scaffolding.    Biomaterials. 2004; 25: 5753-62.    doi:10.1016/j.biomaterials.2004.01.024-   68. Ishihara K, Miyazaki H, Kurosaki T, Nakabayashi N. Improvement    of blood compatibility on cellulose dialysis membrane. 111.    Synthesis and performance of water-soluble cellulose grafted with    phospholipid polymer as coating material on cellulose dialysis    membrane. J Biomed Mater Res. 1995; 29: 181-188.-   69. Bäckdahl H, Helenius G, Bodin A, Nannmark U, Johansson B R,    Risberg B, et al.

Mechanical properties of bacterial cellulose and interactions withsmooth muscle cells. Biomaterials. 2006; 27: 2141-9.doi:10.1016/j.biomaterials.2005.10.026

-   70. Svensson a, Nicklasson E, Harrah T, Panilaitis B, Kaplan D L,    Brittberg M, et al. Bacterial cellulose as a potential scaffold for    tissue engineering of cartilage. Biomaterials. 2005; 26: 419-31.    doi:10.1016/j.biomaterials.2004.02.049-   71. Helenius G, Bäckdahl H, Bodin A, Nannmark U, Gatenholm P,    Risberg B. In vivo biocompatibility of bacterial cellulose. J Biomed    Mater Res Part A. 2006; 76A: 431-438. doi:10.1002/jbm.a.30570-   72. Tischer PCSF, Sierakowski M R, Westfahl H, Tischer C A.    Nanostructural reorganization of bacterial cellulose by ultrasonic    treatment. Biomacromolecules. 2010; 11: 1217-24.    doi:10.1021/bm901383a-   73. Klemm D, Schumann D, Udhardt U, Marsch S. Bacterial synthesized    cellulose artificial blood vessels for microsurgery. Prog Polym Sci.    2001; 26: 1561-1603.-   74. Klemm D, Heublein B, Fink H P, Bohn A. Cellulose: fascinating    biopolymer and sustainable raw material. Angew Chem Int Ed Engl.    2005; 44: 3358-93. doi:10.1002/anie.200460587-   75. Ishihara K, Nakabayashi N, Fukumoto K A J. Improvement of blood    compatibility on cellulose dialysis membrane. Biomaterials. 1992;    13: 145-149.-   76. Gibson L J. The hierarchical structure and mechanics of plant    materials. J R Soc Interface. 2012; 9: 2749-2766.    doi:10.1098/rsif.2012.0341-   77. Derda R, Laromaine A, Mammoto A, Tang S K Y, Mammoto T, Ingber D    E, et al. Paper-supported 3D cell culture for tissue-based    bioassays. PNAS. 2009; 106: 18457-62. doi:10.1073/pnas.0910666106-   78. Bhattacharya M, Malinen M M, Lauren P, Lou Y-RR, Kuisma S W,    Kanninen L, et al. Nanofibrillar cellulose hydrogel promotes    three-dimensional liver cell culture. J Control Release. Elsevier B.    V.; 2012; 164: 291-298. doi:10.1016/j.jconre1.2012.06.039-   79. Brown E E, Hu D, Abu Lail N, Zhang X. Potential of    Nanocrystalline Cellulose-Fibrin Nanocomposites for Artificial    Vascular Graft Applications. Biomacromolecules. American Chemical    Society; 2013; 14: 1063-1071. doi:10.1021/bm3019467-   80. Dugan J M, Collins R F, Gough J E, Eichhorn S J. Oriented    surfaces of adsorbed cellulose nanowhiskers promote skeletal muscle    myogenesis. Acta Biomater. 2013; 9: 4707-15. doi:    10.1016/j.actbio.2012.08.050-   81. Lin N, Dufresne A. Nanocellulose in biomedicine: Current status    and future prospect. Eur Polym J. Elsevier Ltd; 2014; 59: 302-325.    doi:10.1016/j.eurpolymj.2014.07.025-   82. Nimeskern L, Hector M A, Sundberg J, Gatenholm P, Muller R, Stok    K S. Mechanical evaluation of bacterial nanocellulose as an implant    material for ear cartilage replacement. J Mech Behav Biomed Mater.    2013; 22: 12-21. Available:    http://resolver.scholarsportal.info/resolve/17516161/v22icomplete/12_meobnaimfecr.xml-   83. Lu Y, Tekinalp H L, Eberle C C, Peter W, Naskar A K, Ozcan S.    Nanocellulose in polymer composites and biomedical applications.    TAPPI J. TECH ASSOC PULP PAPER IND INC, 15 TECHNOLOGY PARK SOUTH,    NORCROSS, Ga. 30092 USA; 2014; 13: 47-54. Available:    http://apps.webofknowledge.com/full_record.do?product=WOS&search    mode=CitingArticles&    qid=10&SID=2Aza7k6KmLMONuVr81Z&page=l&doc=9&cacheurlFromRightClick=no-   84. Trindade R, Albrektsson T, Tengvall P, Wennerberg A. Foreign    Body Reaction to Biomaterials: On Mechanisms for Buildup and    Breakdown of Osseointegration. Clin Implant Dent Relat Res. 2014;    1-12. doi:10.1111/cid.12274-   85. Onuki Y, Bhardwaj U, Papadimitrakopoulos F, Burgess D J. A    review of the biocompatibility of implantable devices: current    challenges to overcome foreign body response. J diabetes Sci    Technol. 2008; 2: 1003-1015. doi:10.1016/50091-679X(07)83003-2-   86. Anderson J M, Rodriguez A, Chang D T. Foreign body reaction to    biomaterials. Semin Immunol. 2008; 20: 86-100.    doi:10.1016/j.smim.2007.11.004-   87. Jones K S. Effects of biomaterial-induced inflammation on    fibrosis and rejection. Semin Immunol. 2008; 20: 130-136.    doi:10.1016/j.smim.2007.11.005-   88. Nilsson B, Ekdahl K N, Mollnes T E, Lambris J D. The role of    complement in biomaterial-induced inflammation. Mol Immunol. 2007;    44: 82-94. doi:10.1016/j.molimm.2006.06.020-   89. Motegi K, Nakano Y, Namikawa A. Relation between cleavage lines    and scar tissues. J Maxillofac Surg. 1984; 12: 21-8. Available:    http://www.ncbi.nlm.nih.gov/pubmed/6583292-   90. Rickert D, Moses M A, Lendlein A, Kelch S, Franke R-P. The    importance of angiogenesis in the interaction between polymeric    biomaterials and surrounding tissue. Clin Hemorheol Microcirc. 2003;    28: 175-81. Available: http://www.ncbi.nlm.nih.gov/pubmed/12775899-   91. Beguin P. The biological degradation of cellulose. FEMS    Microbiol Rev. 1994; 13: 25-58. doi:10.1016/0168-6445(94)90099-X-   92. Miyamoto T, Takahashi S, Ito H, Inagaki H, Noishiki Y. Tissue    biocompatibility of cellulose and its derivatives. J Biomed Mater    Res. 1989; 23: 125-133. doi:10.1002/jbm.820230110-   93. Dugan J M, Gough J E, Eichhorn S J. Bacterial Cellulose    Scaffolds and Cellulose Nanowhiskers for Tissue Engineering.    Nanomedicine. 2013; 8: 297-298.-   94. Page H, Flood P, Reynaud E G. Three-dimensional tissue cultures:    current trends and beyond. Cell Tissue Res. 2013; 352: 123-31.    doi:10.1007/s00441-012-1441-5-   95. Behravesh E, Yasko a. W, Engel P S, Mikos a. G. Synthetic    Biodegradable Polymers for Orthopaedic Applications. Clin Orthop    Relat Res. 1999; 367: S118-S129.    doi:10.1097/00003086-199910001-00012-   96. Rai R, Keshavarz T, Roether J, Boccaccini A, Roy I. Medium chain    length polyhydroxyalkanoates, promising new biomedical materials for    the future. Mater Sci Eng. Elsevier B. V.; 2011; 72: 29-47.    doi:10.1016/j.mser.2010.11.002-   97. Wang X. Overview on Biocompatibilities of Implantable    Biomaterials. Adv Biomater Sci Appl Biomed. 2013; 112-154.    doi:http://dx.doi.org/10.5772/53461-   98. Chang H, Wang Y. Cell Responses to Surface and Architecture of    Tissue Engineering Scaffolds. Regen Med Tissue Eng Cells Biomater.    2011;-   99. Sittinger M, Bujia J, Rotter N, Reitzel D, Minuth W W, Burmester    G R. Tissue engineering and autologous transplant formation:    practical approaches with resorbable biomaterials and new cell    culture techniques. Biomaterials. 1996; 17: 237-242.    doi:10.1016/0142-9612(96)85561-X-   100. Puschmann T B, Zandén C, De Pablo Y, Kirchhoff F, Pekna M, Liu    J, et al. Bioactive 3D cell culture system minimizes cellular stress    and maintains the in vivo-like morphological complexity of    astroglial cells. Glia. 2013; 61: 432-40. doi:10.1002/glia.22446-   101. Meinel L, Hofmann S, Karageorgiou V, Kirker-Head C, McCool J,    Gronowicz G, et al. The inflammatory responses to silk films in    vitro and in vivo. Biomaterials. 2005; 26: 147-155.    doi:10.1016/j.biomaterials.2004.02.047-   102. Tones F G, Commeaux S, Troncoso O P. Biocompatibility of    bacterial cellulose based biomaterials. J Funct Biomater. 2012; 3:    864-78. doi:10.3390/jfb3040864-   103. Xiao X, Wang W, Liu D, Zhang H, Gao P, Geng L, et al. The    promotion of angiogenesis induced by three-dimensional porous    beta-tricalcium phosphate scaffold with different interconnection    sizes via activation of PI3K/Akt pathways. Sci Rep. 2015; 5: 9409.    doi:10.1038/srep09409-   104. Cancedda R, Giannoni P, Mastrogiacomo M. A tissue engineering    approach to bone repair in large animal models and in clinical    practice. Biomaterials. 2007; 28: 4240-50.    doi:10.1016/j.biomaterials.2007.06.023-   105. Feng B, Jinkang Z, Zhen W, Jianxi L, Jiang C, Jian L, et al.    The effect of pore size on tissue ingrowth and neovascularization in    porous bioceramics of controlled architecture in vivo. Biomed Mater.    2011; 6: 015007. doi:10.1088/1748-6041/6/1/015007-   106. Andrade F K, Silva J P, Carvalho M, Castanheira E M S, Soares    R, Gama M. Studies on the hemocompatibility of bacterial cellulose.    J Biomed Mater Res. 2011; 98: 554-66. doi:10.1002/jbm.a.33148-   107. McBane J E, Sharifpoor S, Cai K, Labow R S, Santerre J P.    Biodegradation and in vivo biocompatibility of a degradable,    polar/hydrophobic/ionic polyurethane for tissue engineering    applications. Biomaterials. Elsevier Ltd; 2011; 32: 6034-44.    doi:10.1016/j.biomaterials.2011.04.048-   108. Orlando G, Wood K J, Stratta R J, Yoo J J, Atala A, Soker S.    Regenerative medicine and organ transplantation: past, present, and    future. Transplantation. 2011; 91: 1310-7.    doi:10.1097/TP.0b013e318219ebb5-   109. Nakayama K H, Batchelder C A, Lee C I, Tarantal A F.    Decellularized Rhesus Monkey Kidney as a Three-Dimensional Scaffold    for Renal Tissue Engineering. Tissue Eng Part A. 2010; 16.    doi:10.1089/ten.tea.2009.0602-   110. Santerre J P, Woodhouse K, Laroche G, Labow R S. Understanding    the biodegradation of polyurethanes: From classical implants to    tissue engineering materials. Biomaterials. 2005; 26: 7457-7470.    doi: 10.1016/j.biomaterials.2005.05.079-   111. Kim M S, Ahn H H, Shin Y N, Cho M H, Khang G, Lee H B. An in    vivo study of the host tissue response to subcutaneous implantation    of PLGA- and/or porcine small intestinal submucosa-based scaffolds.    Biomaterials. 2007; 28: 5137-43.    doi:10.1016/j.biomaterials.2007.08.014-   112. Andrade F, Alexandre N, Amorim I, Gartner F, Mauricio C, Luis    L, et al. Studies on the biocompatibility of bacterial cellulose. J    Bioact Compat Polym. 2012; 28: 97-112. doi:10.1177/0883911512467643-   113. Czaj a WK, Young D J, Kawecki M, Brown R M. The future    prospects of microbial cellulose in biomedical applications.    Biomacromolecules. 2007; 8: 1-12. doi:10.1021/bm060620d-   114. Watanabe K, Eto Y, Takano S, Nakamori S, Shibai H, Yamanaka S.    A new bacterial cellulose substrate for mammalian cell culture.    Cytotechnology. 1993; 13: 107-114. doi:10.1007/BF00749937-   115. Schumann D A, Wippermann J, Klemm D O, Kramer F, Koth D,    Kosmehl H, et al. Artificial vascular implants from bacterial    cellulose: preliminary results of small arterial substitutes.    Cellulose. 2008; 16: 877-885. doi:10.1007/s10570-008-9264-y-   116. Modulevsky, D. J., Lefebvre, C., Haase, K., Al-Rekabi, Z. and    Pelling, A. E. “Apple Derived Cellulose Scaffolds for 3D Mammalian    Cell Culture.” Plos One, 9, e97835 (2014)-   117. http://ascb.org/apple-does-3d-cell-culture/(Sep. 10th 2014)-   118. Modulevsky, D., Cuerrier, C. M. and Pelling, A. E. “Open Source    Biomaterials for Regenerative Medicine.” BioCoder 8, 17 (2015)-   119. Modulevsky, D. & Pelling, A. E. “DIY Open Source Biomaterials.”    BioCoder 8, 43 (2015).-   120. WO 2012056109-   121. EP 2633032-   122. CA 2815276-   123. US 20130344036-   124. US 2013/0224278-   125. WO 2013/126635-   126. AU 2013/222371-   127. U.S. Pat. No. 5,166,187-   128. WO 2008107384-   129. CN 101404977-   130. CN 103224565

All references cited in this section and elsewhere in this specificationare herein incorporated by reference in their entirety.

What is claimed is:
 1. A scaffold biomaterial comprising adecellularised fungal tissue from which cellular materials and nucleicacids of the tissue are removed, the decellularised fungal tissuecomprising a chitin-based 3-dimensional porous structure, wherein thedecellularised fungal tissue has been decellularised by treatment withsodium dodecyl sulphate (SDS), and wherein residual SDS has been removedby using an aqueous divalent salt solution to precipitate a salt residuecontaining SDS micelles out of the scaffold biomaterial.
 2. The scaffoldbiomaterial of claim 1, wherein the decellularised fungal tissuecomprises a fungal tissue which has been further decellularised bythermal shock, treatment with detergent, osmotic shock, lyophilisation,physical lysing, electrical disruption, or enzymatic digestion, or anycombination thereof.
 3. The scaffold biomaterial of claim 1, whereindH₂O, acetic acid, DMSO, or sonication treatment, or any combinationthereof, has been used to remove the aqueous divalent salt solution,salt residue, and/or SDS micelles.
 4. The scaffold biomaterial of claim1, wherein the divalent salt of the aqueous divalent salt solutioncomprises MgCl₂ or CaCl₂.
 5. The scaffold biomaterial of claim 4,wherein the fungal tissue has been decellularised by treatment with anSDS solution of about 1% or about 0.1% SDS in water, and the residualSDS has been removed using an aqueous CaCl₂ solution at a concentrationof about 100 mM followed by incubation in dH₂O.
 6. The scaffoldbiomaterial of claim 1, wherein the decellularised fungal tissue isprocessed to introduce further architecture and/or is functionalized atleast one free hydroxyl functional group through acylation, alkylation,or other covalent modification, to provide a functionalized scaffoldbiomaterial.
 7. The scaffold biomaterial of claim 6, wherein thedecellularised fungal tissue is processed to introduce microchannels,and/or is functionalized with collagen, a factor for promotingcell-specificity, a cell growth factor, or a pharmaceutical agent. 8.The scaffold biomaterial of claim 1, wherein the fungal tissue is amushroom (Fungi) tissue, or a genetically altered tissue produced viadirect genome modification or through selective breeding to create anadditional fungal architecture which is configured to physically mimic atissue and/or to functionally promote a target tissue effect.
 9. Thescaffold biomaterial of claim 1, further comprising living animal cellsadhered to the chitin-based 3-dimensional porous structure.
 10. Thescaffold biomaterial of claim 9, wherein the living animal cells aremammalian cells.
 11. The scaffold biomaterial of claim 10, wherein theliving animal cells are human cells.